Use of a pneumococcal p4 peptide for enhancing opsonophagocytosis in response to a pathogen

ABSTRACT

Methods for enhancing opsonophagocytosis of a pathogen of interest are disclosed. The disclosed methods include administering to a subject an isolated P4 peptide, which includes the amino acid sequence set forth as SEQ ID NO: 1 and optionally an isolated opsonic antibody or a fragment thereof that specifically binds to an antigen present on the surface of the pathogen of interest. In some examples isolated complement protein or a fragment thereof (for example, a C3a, C3b, iC3b, C3d, C4b, or C5a fragment of a complement protein) is also administered. Compositions containing isolated P4 peptide and one or more isolated opsonic antibodies or a fragment thereof that specifically binds to an antigen present on the surface of a pathogen of interest are also disclosed. In some examples, the compositions also include isolated complement protein or fragment thereof, such as one or more of C3a, C3b, iC3b, C3d, C4b, or C5a.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 13/056,522,filed Jan. 28, 2011, which is the §371 U.S. National Stage ofInternational Application No. PCT/US2009/052384, filed Jul. 31, 2009,which was published in English under PCT Article 21(2), which in turnclaims the benefit of U.S. Provisional Patent Application No. 61/085,208filed on Jul. 31, 2008, all of which are incorporated herein byreference in their entirety.

FIELD

This disclosure relates to compositions and methods for the treatmentand/or prevention of pathogenic infections and specifically to enhancingthe effectiveness of opsonic antibodies in the opsonophagocytosis ofpathogens.

BACKGROUND

Over the past century the development of agents to combat infections,such as viral infections, fungal infections, bacterial infections andthe like, has vastly increased the average lifespan throughout theworld. However, pathogens are increasingly developing ways to avoid orcircumvent existing therapeutics. For example, the widespread use oftraditional antibiotics, such as penicillin and related compounds hasresulted in the development of bacteria that are resistant to thesetraditional antibiotics, as exemplified by the rise of methicillinresistant Staphylococcus aureus (MRSA). Similarly, viral pathogens, suchas HIV, are able to acquire resistance to antivirals within a fewreplication cycles.

To combat the ever-changing landscape of pathogens and emergingresistance to the current therapies, the standard course of action forpharmaceutical companies is to develop an ever-increasing array of smallmolecule therapeutic agents. As an alternative, vaccines have beendeveloped which stimulate the body to fight an infection by elicitingantibody responses to the target pathogen(s).

Antibodies protect against pathogen attack by recognizing and binding toantigens on the pathogen to facilitate the removal or “clearance” of thepathogens by a process called phagocytosis, wherein phagocytic cells(for example neutrophils and macrophages) identify, engulf, andsubsequently destroy the pathogens. However, some pathogens, such ascertain bacteria, can avoid phagocytosis. Bacteria can produce a“capsule” that inhibits phagocyte adherence. Opsonic antibodies overcomethese defenses by binding to the capsule or to other target antigens onthe bacterium, in a process called opsonization. This triggers thecomplement cascade, to produce a set of serum proteins with opsonic andlytic activities. Opsonic antibodies with complement components, such asC3a and C5a, bind the bacteria to make the bacteria extremely attractiveto phagocytes and enhance the rate of clearance from the bloodstream.Recently, researches have exploited opsonic antibodies by purifyingopsonic antibodies and administering these antibodies to subjects inorder to treat infections. While the use of opsonic antibodies has shownsome promises in treating and/or preventing infection by pathogens, theneed exists for enhancing the efficacy of these antibodies, for exampleto reduce the amount of the opsonic antibodies needed to achieve atherapeutically effective result. The methods disclosed herein meetthose needs.

SUMMARY

The disclosed methods relate to enhancing the opsonic properties ofopsonic antibodies to augment opsonophagocytosis of pathogens. Thisenhancement is based on the surprising discovery that P4 peptides, whichinclude the amino acid sequence set forth as SEQ ID NO: 1, increase theability of effector cells to internalize pathogens bound by opsonicantibodies. Because P4 peptides increase the ability of effector cellsto opsonophagocytose pathogens bound by opsonic antibodies in anon-discriminate way, P4 peptides can potentially be used to target anypathogen of interest by using an opsonic antibody specific for anypathogen of interest. In specific examples, a P4 peptide is administeredin conjunction with an opsonic antibody that is specific (for examplespecifically binds) for a selected pathogen of interest. Thus, themethods disclosed herein can be used to inhibit and/or treat aninfection from any pathogen of interest.

The disclosed methods of enhancing opsonophagocytosis of a pathogen ofinterest in a subject include administering to the subject atherapeutically effective amount of an isolated P4 peptide that includesthe amino acid sequence set forth as SEQ ID NO: 1. A therapeuticallyeffective amount of an isolated opsonic antibody or a fragment thereof(or even multiple opsonic antibodies) that specifically binds an antigenpresent on the surface of the pathogen of interest can also beadministered to the subject, for example to target a pathogen ofinterest. By administering the P4 peptide the opsonic activity of theopsonic antibody (whether the opsonic antibody is produced by thesubject or administered to the subject) is increased, thereby enhancingthe opsonophagocytosis of a pathogen of interest. In some examples ofthe disclosed method, isolated complement protein or a fragment thereof(for example, a C3a, C3b, iC3b, C3d, C4b, or C5a fragment of acomplement protein) is also administered to the subject. In someembodiments of the disclosed method antibiotic is also administered tothe subject.

The disclosed methods can be used to enhance the opsonophagocytosis ofany pathogen of interest by using an opsonic antibody that binds to aselected pathogen of interest (or antibodies that bind to severalpathogens of interest), for example bacterial pathogens of interest,viral pathogens of interest, virally infected cells, or fungal pathogensof interest, such as those set forth in the summary of terms. Inspecific examples, a pathogen of interest is Streptococcus pneumoniae,Streptococcus pyogenes, Neisseria meningitides or Staphylococcus aureus,such as methicillin resistant Staphylococcus aureus (MRSA). In someexamples, the medicament is used to enhance the opsonophagocytosis ofany pathogen of interest, for example in the treatment of an infectionfrom a pathogen of interest, such as a bacterial pathogen of interest,viral pathogen of interest, virally infected cells, or fungal pathogenof interest, such as those set forth in the summary of terms.

Also disclosed are compositions, such as therapeutic compositions, foruse in treating and/or inhibiting an infection by a pathogen ofinterest, for use in the manufacture of a medicament, and/or for use asmedicament. The disclosed therapeutic compositions include atherapeutically effective amount of isolated P4 peptide including theamino acid sequence set forth as SEQ ID NO: 1 and a therapeuticallyeffective amount of one or more isolated opsonic antibodies or afragment thereof that specifically binds to an antigen present on thesurface of a pathogen of interest. In some embodiments, the disclosedtherapeutic compositions include a therapeutically effective amount ofisolated complement protein or fragment thereof, such as one or more ofC3a, C3b, iC3b, C3d, C4b, or C5a. In some embodiments the disclosedtherapeutic compositions include a therapeutically effective amount ofan antibiotic.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C is a set of bar graphs demonstrating the P4-mediatedenhancement of opsonophagocytosis in vitro. FIG. 1A is a bar graphshowing the effect of antibody concentration on opsonophagocytosis. At a1:8 dilution of gamma globulin, opsonophagocytic killing (OPK) ofStreptococcus pneumoniae serotype 3 (WU2) increased by 35% compared withcontrol; this effect titrated out with dilution, paralleling the resultsobtained for controls at a 1:32 dilution. FIG. 1B is a bar graph showingthe effect of complement on opsonophagocytosis. Baby rabbit complementwas required for in vitro OPK, irrespective of the presence or absenceof P4. The without-complement assay group received heat-inactivatedcomplement (56° C. for 30 minutes). FIG. 1C is a bar graph showing theeffect of P4 concentration on opsonophagocytosis. A gradual increase inthe OPK of S. pneumonia serotype 3 (WU2) over control can be seen withthe increase in P4 concentration. Gamma globulin at a 1:8 dilution wasused as a source of serotype-specific IgG.

FIG. 2A-2C is a set of bar graphs demonstrating the P4-mediatedenhancement of opsonophagocytosis in vitro as determined by flowcytometry. FIG. 2A is a bar graph demonstrating that P4 (100 μg/mL)increased the respiratory burst in HL-60-derived granulocytes. In-housequality control serum (QC2) was used to test the opsonophagocytic uptake(OPU) of OXYBURST®-labeled Streptococcus pneumoniae serotype 23Fcapsular polysaccharide (Ps)-coated beads by granulocytes. The OXYBURST®signal peaked at a serum dilution of 1:3200. FIG. 2B is a bar graphdemonstrating that P4 (100 μg/mL) increased the OPU of S. pneumoniaeserotype 14 Ps-coated beads by freshly isolated granulocytes from humanblood in the presence of QC2. FIG. 2C is a bar graph demonstrating thatP4 (100 μg/mL) increased the OPU of N. meningitidis A Ps-coated beads byHL-60-derived monocytes in the presence of in-house quality-controlserum QC268.

FIG. 3 is a graph demonstrating the conferral of protection againstlethal intranasal Streptococcus pneumoniae serotype 3 (WU2) challenge byP4 with serotype-specific IgG. Intravenous (iv) injection of P4 (100μg/mouse) with gamma globulin (100 μL/mouse) at 72 and 96 hours afterchallenge provided highly significant protection (80%; P<0.001) againstlethal S. pneumoniae WU2 infection, followed in effectiveness by theintraperitoneal (ip) route of administration (60%; P<0.001).

FIG. 4 is a graph demonstrating the survival of mice in varioustreatment arms and of control mice following exposure to S. pneumoniaeserotype 3 (WU2). A single-dose iv injection of a mixture of P4 andgamma globulin (WIG) with ip injection of ceftriaxone (Ceft) providedhighly significant protection (100%; P<0.05) compared to that ofuntreated controls.

FIG. 5 is a graph showing the effects on mice previously rescued (♦)with P4 combination therapy that were reinfected with S. pneumoniaeserotype 3(WU2) on day 28. A single dose of P4, gamma globulin (IVIG),and ceftriaxone was administered 2 days later. All animals (100%) wereprotected.

FIG. 6 is a set of bar graphs showing the results of an in vitroopsonophagocytotic killing assay (OPKA) assay with peripheral blood PMNsisolated from mice 1 and 2 hours postinfection or from uninfected mice.Gamma globulin (IVIG) was used as the source for serotype-specificantibodies. The addition of P4 increased the opsonophagocytic killing ofS. pneumoniae serotype 3 (WU2) by 80% over that by PMNs from controlmice not receiving P4 (P<0.05).

SEQUENCES

The amino acid sequences listed in the accompanying sequence listing areshown using standard the three-letter code for amino acids.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Mar. 25, 2013,and is 4,096 bytes, which is incorporated by reference herein.

SEQ ID NO: 1 is the amino acid sequence of an exemplary P4 peptide.

DETAILED DESCRIPTION I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology canbe found in Benjamin Lewin, Genes VII, published by Oxford UniversityPress, 1999; Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, published by Blackwell Science Ltd., 1994; and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995; and other similarreferences.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “P4 peptide” includes single or pluralpeptides and can be considered equivalent to the phrase “at least a P4peptide.”

As used herein, the term “comprises” means “includes.” Thus, “comprisinga P4 peptide” means “including a P4 peptide” without excluding otherelements.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the invention, thefollowing explanations of terms are provided:

Administration: The introduction of a composition into a subject by achosen route. For example, if the chosen route is intravenous, thecomposition is administered by introducing the composition into a veinof the subject. Similarly, if the route of administration is intranasal,the composition is administered through the nose.

Animal: A living multi-cellular vertebrate or invertebrate organism, acategory that includes, for example, mammals and birds. The term mammalincludes both human and non-human mammals. Similarly, the term “subject”includes both human and veterinary subjects, such as non-human primates.Thus, administration to a subject can include administration to a humansubject. Particular examples of veterinary subjects include domesticatedanimals (such as cats and dogs), livestock (for example, cattle, horses,pigs, sheep, and goats), laboratory animals (for example, mice, rabbits,rats, gerbils, guinea pigs, and non-human primates), as well as birds,reptiles, and fish.

Antibiotic: A compound, composition, or substance that inhibits thegrowth and/or kills bacteria. The term antibiotic can also be used torefer to more than one antibiotic. Examples of antibiotics that can beused with the methods and compositions of this disclosure includewithout limitation, aminoglycosides (such as amikacin, gentamicin,kanamycin, neomycin, netilmicin, streptomycin, tobramycin, andparomomycin); ansamycins (such as geldanamycin, and herbimycin);carbacephems (such as loracarbef, ertapenem, doripenem,imipenem/cilastatin, and meropenem); cephalosporins (such as cefadroxil,cefazolin, cefalotin, cefalexin, cefaclor, cefamandole, cefoxitin,cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone,cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime,ceftriaxone, cefepime, and ceftobiprole); glycopeptides (such asteicoplanin and vancomycin); macrolides (such as azithromycin,clarithromycin, dirithromycin, erythromycin, roxithromycin,troleandomycin, telithromycin, and spectinomycin); monobactams (such asaztreonam); penicillins (such as amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin,meticillin, nafcillin, oxacillin, penicillin, piperacillin, andticarcillin); polypeptides (such as bacitracin, colistin, and polymyxinb); quinolones (such as ciprofloxacin, enoxacin, gatifloxacin,levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin,trovafloxacin, grepafloxacin, and sparfloxacin); sulfonamides (such asmafenide, prontosil (archaic), sulfacetamide, sulfamethizole,sulfanilimide (archaic), sulfasalazine, sulfisoxazole, trimethoprim, andtrimethoprim-sulfamethoxazole); tetracyclines (such as demeclocycline,doxycycline, minocycline, oxytetracycline, and tetracycline); and others(such as arsphenamine, chloramphenicol, clindamycin, lincomycin,ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid,linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin,pyrazinamide, quinupristin/dalfopristin, rifampicin, thiamphenicol, andtinidazole).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies (such as functional antibodies) or a T-cellresponse in a mammal, including compositions that are injected, absorbedor otherwise introduced into a mammal. Examples include, but are notlimited to, peptides, lipids, polysaccharides, and nucleic acidscontaining antigenic determinants, such as those recognized by an immunecell. In some examples, antigens include peptides derived from apathogen of interest. Exemplary pathogens include bacteria, fungi,viruses and parasites. An antigen from a pathogen of interest can beused to produce an opsonic antibody that specifically binds to thepathogen of interest and participate in the opsonophagocytosis of thepathogen.

Antibody: Immunoglobulins and immunologically active portions(“fragments”) thereof, such as molecules that include an antigen bindingsite that specifically binds (immunoreacts with) an antigen. A naturallyoccurring antibody (such as IgG, IgM, and IgA) includes four polypeptidechains, two heavy (H) chains and two light (L) chains interconnected bydisulfide bonds. Examples of immunologically active portions thereof,include, but are not limited to, Fab, Fab′, F(ab′)₂, Fabc and Fvportions. Functional antibodies are antibodies that specifically bind toan antigen, for example an antigen present in the surface of a pathogen,can efficiently allow for complement fixation, and also interact with aneffector cell, wherein the interaction of the antibody and effector cellresults in internalization of the antibody by the effector cell andopsonophagocytosis of the pathogen.

Bacterial pathogen: A bacteria that causes disease (pathogenicbacteria). Examples of pathogenic bacteria against whichopsonophagocytosis can be enhanced in accordance with the disclosedmethods include without limitation any one or more of (or anycombination of) Acinetobacter baumanii, Actinobacillus sp.,Actinomycetes, Actinomyces sp. (such as Actinomyces israelii andActinomyces naeslundii), Aeromonas sp. (such as Aeromonas hydrophila,Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonascaviae), Anaplasma phagocytophilum, Anaplasma marginale, Alcaligenesxylosoxidans, Acinetobacter baumanii, Actinobacillusactinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis,Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillusstearothermophilus), Bacteroides sp. (such as Bacteroides fragilis),Bartonella sp. (such as Bartonella bacilliformis and Bartonellahenselae, Bifidobacterium sp., Bordetella sp. (such as Bordetellapertussis, Bordetella parapertussis, and Bordetella bronchiseptica),Borrelia sp. (such as Borrelia recurrentis, and Borrelia burgdorferi),Brucella sp. (such as Brucella abortus, Brucella canis, Brucellamelintensis and Brucella suis), Burkholderia sp. (such as Burkholderiapseudomallei and Burkholderia cepacia), Campylobacter sp. (such asCampylobacter jejuni, Campylobacter coli, Campylobacter lari andCampylobacter fetus), Capnocytophaga sp., Cardiobacterium hominis,Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci,Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as,Corynebacterium diphtheriae, Corynebacterium jeikeum andCorynebacterium), Clostridium sp. (such as Clostridium perfringens,Clostridium difficile, Clostridium botulinum and Clostridium tetani),Eikenella corrodens, Enterobacter sp. (such as Enterobacter aerogenes,Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli,including opportunistic Escherichia coli, such as enterotoxigenic E.coli, enteroinvasive E. coli, enteropathogenic E. coli,enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenicE. coli) Enterococcus sp. (such as Enterococcus faecalis andEnterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia andEhrlichia canis), Erysipelothrix rhusiopathiae, Eubacterium sp.,Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis,Gemella morbillorum, Haemophilus sp. (such as Haemophilus influenzae,Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae,Haemophilus haemolyticus and Haemophilus parahaemolyticus, Helicobactersp. (such as Helicobacter pylori, Helicobacter cinaedi and Helicobacterfennelliae), Kingella kingii, Klebsiella sp. (such as Klebsiellapneumoniae, Klebsiella granulomatis and Klebsiella oxytoca),Lactobacillus sp., Listeria monocytogenes, Leptospira interrogans,Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp.,Mannheimia hemolytica, Moraxella catarrhalis, Morganella sp., Mobiluncussp., Micrococcus sp., Mycobacterium sp. (such as Mycobacterium leprae,Mycobacterium tuberculosis, Mycobacterium paratuberculosis,Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis,and Mycobacterium marinum), Mycoplasm sp. (such as Mycoplasmapneumoniae, Mycoplasma hominis, and Mycoplasma genitalium), Nocardia sp.(such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardiabrasiliensis), Neisseria sp. (such as Neisseria gonorrhoeae andNeisseria meningitidis), Pasteurella multocida, Plesiomonasshigelloides. Prevotella sp., Porphyromonas sp., Prevotellamelaminogenica, Proteus sp. (such as Proteus vulgaris and Proteusmirabilis), Providencia sp. (such as Providencia alcalifaciens,Providencia rettgeri and Providencia stuartii), Pseudomonas aeruginosa,Propionibacterium acnes, Rhodococcus equi, Rickettsia sp. (such asRickettsia rickettsii, Rickettsia akari and Rickettsia prowazekii,Orientia tsutsugamushi (formerly: Rickettsia tsutsugamushi) andRickettsia typhi), Rhodococcus sp., Serratia marcescens,Stenotrophomonas maltophilia, Salmonella sp. (such as Salmonellaenterica, Salmonella typhi, Salmonella paratyphi, Salmonellaenteritidis, Salmonella cholerasuis and Salmonella typhimurium),Serratia sp. (such as Serratia marcesans and Serratia liquifaciens),Shigella sp. (such as Shigella dysenteriae, Shigella flexneri, Shigellaboydii and Shigella sonnei), Staphylococcus sp. (such as Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus hemolyticus,Staphylococcus saprophyticus), Streptococcus sp. (such as Streptococcuspneumoniae (for example chloramphenicol-resistant serotype 4Streptococcus pneumoniae, spectinomycin-resistant serotype 6BStreptococcus pneumoniae, streptomycin-resistant serotype 9VStreptococcus pneumoniae, erythromycin-resistant serotype 14Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcuspneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae,tetracycline-resistant serotype 19F Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, andtrimethoprim-resistant serotype 23F Streptococcus pneumoniae,chloramphenicol-resistant serotype 4 Streptococcus pneumoniae,spectinomycin-resistant serotype 6B Streptococcus pneumoniae,streptomycin-resistant serotype 9V Streptococcus pneumoniae,optochin-resistant serotype 14 Streptococcus pneumoniae,rifampicin-resistant serotype 18C Streptococcus pneumoniae,penicillin-resistant serotype 19F Streptococcus pneumoniae, ortrimethoprim-resistant serotype 23F Streptococcus pneumoniae),Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes,Group A streptococci, Streptococcus pyogenes, Group B streptococci,Streptococcus agalactiae, Group C streptococci, Streptococcus anginosus,Streptococcus equismilis, Group D streptococci, Streptococcus bovis,Group F streptococci, and Streptococcus anginosus Group G streptococci),Spirillum minus, Streptobacillus moniliformi, Treponema sp. (such asTreponema carateum, Treponema petenue, Treponema pallidum and Treponemaendemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonellasp., Vibrio sp. (such as Vibrio cholerae, Vibrio parahemolyticus, Vibriovulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrioalginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibriometchnikovii, Vibrio damsela and Vibrio furnisii), Yersinia sp. (such asYersinia enterocolitica, Yersinia pestis, and Yersiniapseudotuberculosis) and Xanthomonas maltophilia among others.

Cell: A plant, animal, insect, bacterial, or fungal cell.

Conservative amino acid substitutions providing functionally similaramino acids are well known in the art. The following six groups eachcontain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Not all residue positions within a protein will tolerate an otherwise“conservative” substitution. For instance, if an amino acid residue isessential for a function of the protein, even an otherwise conservativesubstitution may disrupt that activity.

Complement: A plasma protein system involved in immune defense.Following activation by antigen-antibody complexes, complement proteinslyse antigenic cells, attract phagocytic cells, and assist in thedestruction of antigenic cells by opsonophagocytosis. In mammals, thecomplement system is made up of a series of about 25 proteins that workto “complement” the activity of antibodies in destroying bacteria,either by facilitating opsonophagocytosis or by puncturing the bacterialcell membrane. Complement also helps to rid the body of antigen-opsonicantibody complexes, for example by clearance of a pathogen that is boundby the opsonic antibody.

Complement proteins circulate in the blood in an inactive form. When thefirst of the complement substances is triggered-usually by antibodyinterlocked with an antigen. As each component is activated in turn, itacts upon the next in a precise sequence of carefully regulated stepsknown as the “complement cascade.”

Complement fragments (such as C3a, C3b, iC3b, C3d, C4b, or C5a, whichbecome bound to antigen during complement activation) triggeropsonophagocytosis by binding to specific cell-surface receptors (suchas Fc receptors and C3b receptors on neutrophils and macrophages, andC3d receptors on macrophages). In some examples, the activity of aneffector cells for opsonophagocytosis of antibody/antigen complexes isenhanced by the presence of P4 peptides.

Effector cells: Cells capable of binding to antibody/antigen complexesand internalizing such complexes. In particular examples, effector cellsexpress Fc receptors, such as FcγRI, FcγRII and FcγRIII that bind toantibody/antigen complexes and facilitate internalization. In someexamples, effector cells are derived from the serum of an individual(such as peripheral blood leukocytes, PBLs) or from an in vitro culture.Examples of effector cells include, but are not limited to: macrophages,mononuclear phagocytes, natural killer cells, and granulocytes such asneutrophils and eosinophils. In a particular example, the effector cellis a differentiated human promyelocytic leukemia cell, such as adifferentiated HL-60 cell.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, such that theyelicit a specific immune response. An antibody binds a particularantigenic epitope, such as an epitope of on the surface of a pathogen.

Exogenous: A substance, such as an isolated opsonic antibody orcomplement protein of fragment thereof, that is obtained from a sourceother than the subject to which it is administered. For example, when anexogenous isolated opsonic antibody is administered to a subjectaccording to the methods and compositions described herein, thatisolated antibody is not obtained, for example isolated from the samesubject to whom it is administered.

Fungal pathogen: A fungus that causes disease. Examples of fungalpathogens for which opsonophagocytosis can be enhanced in accordancewith the disclosed methods include without limitation Trichophytonrubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis,Pityrosporum orbiculare (Malassezia furfur), Candida sp. (such asCandida albicans), Aspergillus sp. (such as Aspergillus fumigatus,Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp. (such asCryptococcus neoformans, Cryptococcus gattii, Cryptococcus laurentii andCryptococcus albidus), Histoplasma sp. (such as Histoplasma capsulatum),Pneumocystis sp. (such as Pneumocystis jirovecii), and Stachybotrys(such as Stachybotrys chartarum) among others.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as an infection with a pathogen, for example a bacterial,fungal or viral pathogen. “Treatment” refers to a therapeuticintervention that ameliorates a sign or symptom of a disease orpathological condition after it has begun to develop. The term“ameliorating,” with reference to a disease or pathological condition,refers to any observable beneficial effect of the treatment. Thebeneficial effect can be evidenced, for example, by a delayed onset ofclinical symptoms of the disease in a susceptible subject, a reductionin severity of some or all clinical symptoms of the disease, a slowerprogression of the disease, an improvement in the overall health orwell-being of the subject, or by other parameters well known in the artthat are specific to the particular disease. A “prophylactic” treatmentis a treatment administered to a subject who does not exhibit signs of adisease or exhibits only early signs for the purpose of decreasing therisk of developing pathology.

Isolated: An “isolated” biological component (such as a protein, forexample P4 peptide, antibody or complement protein) has beensubstantially separated or purified away from other biologicalcomponents in which the component naturally occurs, such as otherchromosomal and extrachromosomal DNA, RNA, and proteins. Proteins orpeptides that have been “isolated” include proteins purified by standardpurification methods. The term also embraces proteins or peptidesprepared by recombinant expression in a host cell as well as chemicallysynthesized proteins or peptides. Isolated does not require absolutepurity, and can include protein or peptide molecules that are at least50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even100% isolated.

Opsonin: A molecule that becomes attached to the surface of a pathogen,such as a bacterial, fungal or viral pathogen, that be recognized bysurface receptors of neutrophils and macrophages and that increases theefficiency of phagocytosis of the microbe. Opsonins include IgGantibodies, which are recognized by the Fcγ receptor on phagocytes, andfragments of complement proteins, which are recognized by CR1 (CD35) andby the leukocyte integrin Mac-1.

Opsonophagocytosis: The process of attaching opsonins to microbialsurfaces to target the microbes for phagocytosis by effector cells (suchas macrophages and monocytes) in the presence of specific serumopsonins. Opsonins include any substance that binds to particulateantigens and induces their phagocytosis by effector cells. Exemplaryopsonins include opsonizing antibodies (IgM, IgG1, IgG2, IgG3 and IgAimmunoglobulins specific for the antigen) and certain complementfragments (C3a, C3b, iC3b, C3d, C4b, or C5a, which become bound to theantigen during complement activation), both of which triggerphagocytosis by binding to specific cell-surface receptors (such as Fcreceptors and C3b receptors on neutrophils and macrophages, and C3dreceptors on macrophages). In some examples, the activity of an effectorcells for opsonophagocytosis of antibody/antigen complexes is enhancedby the presence of P4 peptides.

Peptide: Any compound composed of amino acids, amino acid analogs,chemically bound together. Peptide as used herein includes oligomers ofamino acids, amino acid analog, or small and large peptides, includingpolypeptides or proteins. Any chain of amino acids, regardless of lengthor post-translational modification (such as glycosylation orphosphorylation). “Peptide” applies to amino acid polymers to naturallyoccurring amino acid polymers and non-naturally occurring amino acidpolymer as well as in which one or more amino acid residue is anon-natural amino acid, for example an artificial chemical mimetic of acorresponding naturally occurring amino acid. In some embodiments, thepeptide is a P4 peptide, which can include but is not limited to any ofthe modifications described herein. A “residue” refers to an amino acidor amino acid mimetic incorporated in a polypeptide by an amide bond oramide bond mimetic. A peptide has an amino terminal (N-terminal) end anda carboxy terminal (C-terminal) end. “Peptide” is used interchangeablywith polypeptide or protein, and is used interchangeably herein to referto a polymer of amino acid residues.

Amino acids generally are chemically bound together via amide linkages(CONH). Additionally, amino acids may be bound together by otherchemical bonds. For example, linkages for amino acids or amino acidanalogs can include CH₂NH—CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans),—COCH₂—, CH(OH)CH₂—, and —CHH₂SO— (These and others can be found inSpatola, in Chemistry and Biochemistry of Amino Acids, Peptides, andProteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983);Spatula, A. F., Vega Data (March 1983), Vol. 1, Issue 3, PeptideBackbone Modifications (general review); Morley, Trends Pharm Sci pp.463-468, 1980; Hudson, et al., Int J Pept Prot Res 14:177-185, 1979;Spatola et al. Life Sci 38:1243-1249, 1986; Harm J. Chem. Soc PerkinTrans. 1307-314, 1982; Almquist et al. J. Med. Chem. 23:1392-1398, 1980;Jennings-White et al. Tetrahedron Lett 23:2533, 1982; Holladay et al.Tetrahedron. Lett 24:4401-4404, 1983; and Hruby Life Sci 31:189-199,1982.

Peptides may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedproteins, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁-C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁-C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the protein,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁-C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the protein side chains may be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the protein side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁-C₁₆ alkyl, C₁-C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the protein sidechains can be extended to homologous C₂-C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the proteins to selectand provide conformational constraints to the structure that result inenhanced stability.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use with the disclosed methods are conventional carriers.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition, 1975, describes compositions andformulations suitable for pharmaceutical delivery of peptides andproteins, such as P4 peptides, opsonic antibodies and complementproteins, or fragments thereof.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol, or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, preservatives, and pH buffering agents and the like, for examplesodium acetate or sorbitan monolaurate.

Pharmaceutical agent or drug: A chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect when properlyadministered to a subject.

Serotype: The genotype of a unicellular organism, such as a bacterium,as defined by antisera against antigenic determinants expressed on thesurface. Can also refer to the antigens themselves.

Specifically bind: When referring to an opsonin (such as an opsonicantibody), refers to a binding reaction which is determinative of thepresence of a target protein, peptide, or polysaccharide in the presenceof a heterogeneous population of proteins and other biologics. Thus,under designated conditions, an antibody binds preferentially to aparticular target protein, peptide or polysaccharide (such as an antigenpresent on the surface of a pathogen, for example bacterial capsularpolysaccharide) and do not bind in a significant amount to otherproteins or polysaccharides present in the sample or subject.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn, and tblastx. Blastn is used tocompare nucleic acid sequences, while blastp is used to compare aminoacid sequences. Additional information can be found at the NCBI website.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresent in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a peptide sequence that has 1166matches when aligned with a test sequence having 1554 nucleotides is75.0 percent identical to the test sequence (1166÷1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer.

Therapeutically effective amount: A quantity of a specific substance(for example P4 peptide, opsonic antibody, antibiotic and/or complementprotein) sufficient to achieve a desired effect in a subject beingtreated. For instance, this can be the amount necessary to inhibit ortreat an infection by a pathogen, such as an infection by a bacterialpathogen. When administered to a subject, a dosage will generally beused that will achieve target tissue concentrations shown to achieve adesired in vitro effect.

A therapeutically effective amount of a substance, such as P4 peptide,opsonic antibody and/or complement protein can be administered in asingle dose, or in several doses, for example daily, during a course oftreatment. However, the effective amount of a composition will bedependent on the compound or peptide applied, the subject being treated,the severity and type of the affliction, and the manner ofadministration of the composition. For example, a therapeuticallyeffective amount of composition can vary from about 0.01 mg/kg bodyweight to about 1 g/kg body weight.

Virus: A microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of nucleic acid surroundedby a protein coat, and has the ability to replicate only inside a livingcell. “Viral replication” is the production of additional virus by theoccurrence of at least one viral life cycle. A virus may subvert thehost cells' normal functions, causing the cell to behave in a mannerdetermined by the virus. For example, a viral infection may result in acell producing a cytokine, or responding to a cytokine, when theuninfected cell does not normally do so. In some examples, a virus is apathogen. Specific examples of viral pathogens for whichopsonophagocytosis can be enhanced in accordance with the disclosedmethods include, without limitation; Arenaviruses (such as Guanaritovirus, Lassa virus, Junin virus, Machupo virus and Sabia),Arteriviruses, Roniviruses, Astroviruses, Bunyaviruses (such asCrimean-Congo hemorrhagic fever virus and Hantavirus), Barnaviruses,Birnaviruses, Bornaviruses (such as Borna disease virus), Bromoviruses,Caliciviruses, Chrysoviruses, Coronaviruses (such as Coronavirus andSARS), Cystoviruses, Closteroviruses, Comoviruses, Dicistroviruses,Flaviruses (such as Yellow fever virus, West Nile virus, Hepatitis Cvirus, and Dengue fever virus), Filoviruses (such as Ebola virus andMarburg virus), Flexiviruses, Hepeviruses (such as Hepatitis E virus),human adenoviruses (such as human adenovirus A-F), human astroviruses,human BK polyomaviruses, human bocaviruses, human coronavirus (such as ahuman coronavirus HKU1, NL63, and OC43), human enteroviruses (such ashuman enterovirus A-D), human erythrovirus V9, human foamy viruses,human herpesviruses (such as human herpesvirus 1 (herpes simplex virustype 1), human herpesvirus 2 (herpes simplex virus type 2), humanherpesvirus 3 (Varicella zoster virus), human herpesvirus 4 type 1(Epstein-Barr virus type 1), human herpesvirus 4 type 2 (Epstein-Barrvirus type 2), human herpesvirus 5 strain AD169, human herpesvirus 5strain Merlin Strain, human herpesvirus 6A, human herpesvirus 6B, humanherpesvirus 7, human herpesvirus 8 type M, human herpesvirus 8 type Pand Human Cyotmegalovirus), human immunodeficiency viruses (HIV) (suchas HIV 1 and HIV 2), human metapneumoviruses, human papillomaviruses(such as human papillomavirus-1, human papillomavirus-18, humanpapillomavirus-2, human papillomavirus-54, human papillomavirus-61,human papillomavirus-c and 90, human papillomavirus RTRX7, humanpapillomavirus type 10, human papillomavirus type 101, humanpapillomavirus type 103, human papillomavirus type 107, humanpapillomavirus type 16, human papillomavirus type 24, humanpapillomavirus type 26, human papillomavirus type 32, humanpapillomavirus type 34, human papillomavirus type 4, humanpapillomavirus type 41, human papillomavirus type 48, humanpapillomavirus type 49, human papillomavirus type 5, humanpapillomavirus type 50, human papillomavirus type 53, humanpapillomavirus type 60, human papillomavirus type 63, humanpapillomavirus type 6b, human papillomavirus type 7, humanpapillomavirus type 71, human papillomavirus type 9, humanpapillomavirus type 92, and human papillomavirus type 96), humanparainfluenza viruses (such as human parainfluenza virus 1-3), humanparechoviruses, human parvoviruses (such as human parvovirus 4 and humanparvovirus B 19), human respiratory syncytial viruses, humanrhinoviruses (such as human rhinovirus A and human rhinovirus B), humanspumaretroviruses, human T-lymphotropic viruses (such as humanT-lymphotropic virus 1 and human T-lymphotropic virus 2), Human polyomaviruses, Hypoviruses, Leviviruses, Luteoviruses, Lymphocyticchoriomeningitis viruses (LCM), Marnaviruses, Narnaviruses, Nidovirales,Nodaviruses, Orthomyxoviruses (such as Influenza viruses),Partitiviruses, Paramyxoviruses (such as Measles virus and Mumps virus),Picornaviruses (such as Poliovirus, the common cold virus, and HepatitisA virus), Potyviruses, Poxviruses (such as Variola and Cowpox),Sequiviruses, Reoviruses (such as Rotavirus), Rhabdoviruses (such asRabies virus), Rhabdoviruses (such as Vesicular stomatitis virus,Tetraviruses, Togaviruses (such as Rubella virus and Ross River virus),Tombusviruses, Totiviruses, Tymoviruses, Noroviruses, bovineherpesviruses including Bovine Herpesvirus (BHV) and malignant catarrhalfever virus (MCFV), among others. In some examples, a cell infected witha virus is opsonophagocytosed

II. Overview of Several Embodiments

This disclosure is related to methods of enhancing the opsonic responseto pathogens and compositions for use in targeting a pathogen ofinterest for opsonophagocytosis. Opsonophagocytosis is the binding (oropsonization) of antibodies and complement or complement components tothe pathogen and the subsequent uptake of the infectious agent byeffector cells via the binding of the effector cells to theantibody/antigen complex.

During a protective immune response, functional antibodies are generatedthat bind to the infectious agents and also provide a means for uptakeand clearance by effector cells. Functional antibodies (opsonicantibodies) that specifically bind an antigen on the surface of apathogen of interest can be purified and administered to a subject totreat and/or inhibit an infection in the subject by targeting thepathogen of interest for opsonization by the subjects own effectorcells. While such therapies appear promising, typically large doses ofthe opsonic antibody must be administered to reach the desired effect ofpathogen clearance. Thus, the need exists for methods of enhancing theopsonic response for opsonic antibodies.

To meet this need, disclosed herein are methods of using P4 peptides toenhance the opsonic response to opsonic antibodies by effector cellswhen administered in conjunction with opsonic antibodies that targetpathogens of interest by specifically binding antigen on their surface.The P4 peptides are derived from strand 7, α-helix 12, and strand 8 ofthe Streptococcus pneumoniae PsaA protein, but contain point mutationsrelative to the native sequence of the PsaA protein to increase theisoelectric point and enhance binding. The discovery that P4 peptidesenhanced the opsonization of pathogens by effector cells, as disclosedherein, was particularly surprising and unexpected considering that theP4 peptides were initially developed to inhibit the internalization ofStreptococcus pneumoniae by nasopharyngeal cells (see InternationalPatent Publication WO 2006/127020). Furthermore, as disclosed herein, athas also been discovered that the coadministration of P4 peptide withantibiotics synergistically enhances the effectiveness of theantibiotics. Because of this synergistic behavior, it is possible to uselower doses of antibiotic when combined with P4 peptide, while stillmaintaining the effectiveness of the antibiotic.

A Methods of Treatment

Methods of enhancing opsonophagocytosis of a pathogen of interest in asubject are disclosed, for example to enhance the opsonophagocytosis ofa pathogen of interest for the purpose of inhibiting and/or treating aninfection in a subject from the pathogen of interest. The pathogen ofinterest can be any of the bacterial, viral or fungal pathogensdiscussed in the forgoing summary of terms. In particular examples themethod is used to enhance the opsonophagocytosis in a subject who isinfected with (or at risk of being infected with) a pathogen, (such as aviral, bacterial or fungal pathogen). In particular examples thepathogen is a bacterial pathogen, such as Streptococcus (such asStreptococcus Pneumoniae), Staphylococcus (such as Staphylococcusaureus), or Meningococcus (such as Neisseria meningitides). In someexamples, a subject is selected for treatment that has or is at risk fordeveloping an infection by a pathogen, (such as a viral, bacterial orfungal pathogen). In some examples, a subject is selected that has or isat risk of a Streptococcus infection (such as a Streptococcus Pneumoniaeinfection). In some examples, a subject is selected that has or is atrisk of a Staphylococcus infection (such as a Staphylococcus aureusinfection). In some examples, a subject is selected that has or is atrisk of a Meningococcus infection (such as a Neisseria meningitidesinfection). In some examples a subject selected for treatment is notinfected with a pathogen expressing Pneumococcal surface adhesin A(PsaA) protein, for example the selected subject is not infected withStreptococcus Pneumoniae. The methods include administering to thesubject a therapeutically effective amount of isolated P4 peptide thatincludes an amino acid sequence that is at least 95% identical, such asat least 96% at least 97% at least 99% at least 99% identical or even100% identical, to the amino acid sequence set forth asLFVESSVKRRPMKTVSQDTNIPIYAQIF (SEQ ID NO: 1), and optionally atherapeutically effective amount of one or more isolated opsonicantibodies or a fragment thereof that specifically bind an antigenpresent on the surface of the pathogen of interest (for example abacterial, viral or fungal pathogen of interest listed above). In someexamples a P4 peptide is between about 27 and about 200 amino acids inlength, such as no more than 28, 29, 30, 40, 50, 60, 70, 80, 90, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids inlength or even longer, for example 27-50, 40-60, 50-70, 60-80, 70-90,80-100, 90-110, 100-120, 110-130, 120-140, 130-140, 140-160, 150-170,160-180, 170-190, or 180-200, amino acids in length. In some examples, aP4 peptide consists of the amino acid sequence set forth as SEQ IDNO: 1. In some examples, a subject is administered P4 peptide. In someexamples, a subject is administered P4 peptide in conjunction with oneor more isolated opsonic antibodies or a fragment thereof thatspecifically bind an antigen present on the surface of the pathogen ofinterest. In some examples, a subject is administered P4 peptide thathas cells infected with a virus, for example to enhance theopsonophagocytosis of the cells expressing cell surface proteins fromthe viral pathogen of interest. In some examples, the P4 peptideincludes an amino acid sequence that has no more than one or two aminoacid changes from the amino acid sequence set forth as SEQ ID NO: 1, forexample conservative substitutions. Changes in amino acid sequence canbe utilized will still render the resultant peptide capable of enhancingopsonophagocytosis of a pathogen of interest are contemplated forexample by conservative substitution. Also contemplated are fusionproteins that include a P4 peptide fused to a heterologous amino acidsequence. In some examples, the P4 peptide is lipidated, for examplelipidated with a palmitic acid and the like. Exemplary pharmaceuticalcompositions are described below in section B. Various modes ofadministration of the pharmaceutical compositions of this disclosure arecontemplated (see section B below).

The administration of the P4 peptide enhances the subject's ability (andspecifically the ability of effector cells of the subject) toopsonophagocytose the pathogen of interest that is specifically bound byan opsonic antibody or fragment thereof, for example an opsonic antibodyproduced by the subject (for example a subject infected with a pathogen)and/or an isolated opsonic antibody administered to the subject. In someexamples, an opsonic antibody or fragment thereof and the P4 peptide isadministered to a subject. The administration of an opsonic antibody orfragment thereof and the P4 peptide can occur in any order or evensimultaneously, for example by co-administration as a singlepharmaceutical preparation, or as multiple preparations, such as apharmaceutical composition that contains a therapeutically effectiveamount of P4 peptide and a composition that contains a therapeuticallyeffective amount of an opsonic antibody or fragment thereof thatspecifically binds a pathogen of interest (or even multiple opsonicantibodies, for example multiple opsonic antibodies that eachspecifically bind a single pathogen of interest, or multiple opsonicantibodies where each opsonic antibody specifically binds a differentpathogen of interest, or multiple serotypes of a single pathogen ofinterest, or any combination thereof) or even a composition thatcontains both a therapeutically effective amount of P4 peptide and atherapeutically effective amount of an opsonic antibody or fragmentthereof (or multiple opsonic antibodies).

As disclosed herein the administration of the P4 peptide in conjunctionwith an antibiotic increases the effectiveness of the antibiotic, forexample allowing a lower dose to be used and/or increasing bacterialclearance. In general any antibiotic can be used with the disclosedmethods. Examples of antibiotics that can be used include but are notlimited to aminoglycosides (such as amikacin, gentamicin, kanamycin,neomycin, netilmicin, streptomycin, tobramycin, and paromomycin);ansamycins (such as geldanamycin, and herbimycin); carbacephems (such asloracarbef, ertapenem, doripenem, imipenem/cilastatin, and meropenem);cephalosporins (such as cefadroxil, cefazolin, cefalotin, cefalexin,cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime,cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, andceftobiprole); glycopeptides (such as teicoplanin and vancomycin);macrolides (such as azithromycin, clarithromycin, dirithromycin,erythromycin, roxithromycin, troleandomycin, telithromycin, andspectinomycin); monobactams (such as aztreonam); penicillins (such asamoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin,dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin,oxacillin, penicillin, piperacillin, and ticarcillin); polypeptides(such as bacitracin, colistin, and polymyxin b); quinolones (such asciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin,moxifloxacin, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, andsparfloxacin); sulfonamides (such as mafenide, prontosil (archaic),sulfacetamide, sulfamethizole, sulfanilimide (archaic), sulfasalazine,sulfisoxazole, trimethoprim, and trimethoprim-sulfamethoxazole);tetracyclines (such as demeclocycline, doxycycline, minocycline,oxytetracycline, and tetracycline); and others (such as arsphenamine,chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin,fusidic acid, furazolidone, isoniazid, linezolid, metronidazole,mupirocin, nitrofurantoin, platensimycin, pyrazinamide,quinupristin/dalfopristin, rifampicin, thiamphenicol, and tinidazole).In some examples, an antibiotic (or more than one antibiotic) and the P4peptide is administered to a subject. The administration of anantibiotic or fragment thereof and the P4 peptide can occur in any orderor even simultaneously, for example by co-administration as a singlepharmaceutical preparation, or as multiple preparations, such as apharmaceutical composition that contains a therapeutically effectiveamount of P4 peptide and a composition that contains a therapeuticallyeffective amount of an antibiotic that is specific for a pathogen ofinterest. In some examples, an opsonic antibody or fragment thereof, P4peptide and antibiotic is administered to a subject. The administrationof an opsonic antibody or fragment thereof, the P4 peptide and theantibiotic can occur in any order or even simultaneously, for example byco-administration as a single pharmaceutical preparation, or as multiplepreparations, such as a pharmaceutical composition that contains atherapeutically effective amount of P4 peptide, a composition thatcontains a therapeutically effective amount of an antibiotic specificfor a pathogen of interest, and a composition that contains atherapeutically effective amount of an opsonic antibody or fragmentthereof that specifically binds a pathogen of interest (or even multipleopsonic antibodies, for example multiple opsonic antibodies that eachspecifically bind a single pathogen of interest, or multiple opsonicantibodies where each opsonic antibody specifically binds a differentpathogen of interest, or multiple serotypes of a single pathogen ofinterest, or any combination thereof) or even a composition thatcontains a therapeutically effective amount of P4 peptide and atherapeutically effective amount of an opsonic antibody or fragmentthereof (or multiple opsonic antibodies) and a therapeutically effectiveamount of an antibiotic specific for a pathogen of interest.

Complement proteins and fragments thereof assist in opsonophagocytosisof pathogens by binding to opsonic antibodies and facilitating theopsonization by effector cells. Thus, in some examples, the subject isalso administered a pharmaceutically effective amount of an isolatedcomplement protein or fragment thereof, such as one or more of C3a, C3b,iC3b, C3d, C4b, or C5a. In some examples a subject is selected that hasa complement defect for administration of a pharmaceutically effectiveamount of an isolated complement protein or fragment thereof. In certainexamples the therapeutically effective amount of the isolated P4 peptideis administered by an intranasal route and/or an intravenous route. Insome embodiments, the therapeutically effective amount of the opsonicantibody or fragment thereof is administered by an intranasal routeand/or an intravenous route. In some examples, the pharmaceuticallyeffective amount of an isolated complement protein or a fragmentthereof, such as one or more of C3a, C3b, iC3b, C3d, C4b, or C5a, isadministered by an intranasal route and/or an intravenous route.

Because P4 peptides are not specific for any single pathogen, the P4peptides and therapeutic compositions of this disclosure can beformulated to enhance the opsonophagocytosis of any pathogen of interestby providing an opsonic antibody or fragment thereof that targets anypathogen of interest, for example by providing an opsonic antibody or afragment thereof (such as a therapeutically effective amount of anopsonic antibody or a fragment thereof) that specifically binds anantigen present on the surface of the pathogen of interest, or providingP4 peptide to a subject that is producing opsonic antibodies, forexample a subject that is infected, or has been infected with apathogen. Methods of producing opsonic antibodies are given below insection C. In some examples, the pathogen of interest is a bacterialpathogen and a therapeutically effective amount of an opsonic antibodythat specifically binds the bacterial pathogen is provided. In certainexamples, the pathogen of interest is Streptococcus pneumoniae. In otherexamples, the pathogen of interest is Neisseria meningitides. In stillother examples, the pathogen of interest is Staphylococcus aureus, suchas is methicillin resistant Staphylococcus aureus (MRSA). In someexamples, the pathogen of interest is a viral pathogen and atherapeutically effective amount of an opsonic antibody thatspecifically binds the viral pathogen or a cell infected with the viralpathogen is provided. In some examples, the pathogen of interest is afungal pathogen and a therapeutically effective amount of an opsonicantibody that specifically binds the fungal pathogen is provided.

B. Therapeutic Compositions

The P4 peptide can be administered in vitro, ex vivo or in vivo to acell or subject. It is desirable to prepare P4 peptides as apharmaceutical composition appropriate for the intended application, forexample to inhibit or treat a pathogenic infection, such as an infectionby a pathogen discussed in the foregoing summary of terms. Accordingly,methods for making a medicament or pharmaceutical composition containinga P4 peptide (and in some cases an opsonic antibody or a fragmentthereof, antibiotic, and/or complement protein or a fragment thereof)are included herein. P4 peptides can be prepared for administrationalone or with other active ingredients, such as antibiotics (for examplethe antibiotics described in Section A above) and/or other proteins,such as with an opsonic antibody, antibiotic (or even multipleantibiotics) and/or complement protein (or even multiple opsonicantibodies that are specific for different pathogens (cells infectedwith pathogens) and/or complement proteins, or fragments thereof). Insome examples, a therapeutic composition includes P4 peptide. In someexamples, a therapeutic composition includes an opsonic antibody. Insome examples, a therapeutic composition includes an antibiotic. In someexamples, a therapeutic composition includes an opsonic antibody and aP4 peptide. In some examples, a therapeutic composition includes anopsonic antibody, a P4 peptide and an antibiotic. In some examples, atherapeutic composition includes a complement protein or fragmentthereof. In some examples, a therapeutic composition includes acomplement protein or fragment thereof and a P4 peptide. In someexamples, a therapeutic composition includes a complement protein orfragment thereof, an antibiotic and a P4 peptide. In some examples, atherapeutic composition includes a complement protein or fragmentthereof, an opsonic antibody, an antibiotic and a P4 peptide. In someexamples, a therapeutic composition includes a complement protein orfragment thereof and an opsonic antibody. When P4 peptide and opsonicantibody, and/or antibiotic and/or complement protein is administered toa subject, the administration can be concurrent or sequential.Sequential administration of the P4 peptide and opsonic antibody and/orantibiotic and/or complement protein can be separated by any amount oftime so long as the administration of P4 peptide enhances the opsonicactivity of the opsonic antibody. Multiple administrations of thecompositions described herein are also contemplated.

In some embodiments, a disclosed therapeutic composition includes atherapeutically effective amount of isolated P4 peptide that includes anamino acid sequence that is at least 95% identical such as at least 96%,at least 97%, at least 98%, at least 99%, or even 100% identical to setforth as SEQ ID NO: 1 and optionally a therapeutically effective amountof one or more isolated opsonic antibodies or a fragment thereof thatspecifically binds to an antigen present on the surface of a pathogen ofinterest. In some examples, the therapeutic composition also includes atherapeutically effective amount of an antibiotic, or even more than oneantibiotic. In some examples, the therapeutic composition also includesa therapeutically effective amount of an isolated complement protein orfragment thereof, such as one or more of C3a, C3b, iC3b, C3d, C4b, orC5a.

Typically, preparation of a pharmaceutical composition (for use as amedicament or in the manufacture of a medicament) entails preparing apharmaceutical composition that is essentially free of pyrogens, as wellas any other impurities that could be harmful to humans or animals.Typically, the pharmaceutical composition contains appropriate salts andbuffers to render the components of the composition stable and allow theP4 peptide to interact with cells of a subject.

Administration of therapeutic compositions can be by any common route aslong as the target tissue is available via that route. This includesoral, nasal (such as intranasal), ocular, buccal, enteral, intravitreal,or other mucosal (such as rectal or vaginal) or topical administration.Alternatively, administration will be by orthotopic, intradermalsubcutaneous, intramuscular, parenteral, intraperitoneal, or intravenousinjection routes. Such pharmaceutical compositions are usuallyadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

Therapeutic compositions can be provided as parenteral compositions,such as for injection or infusion. Such compositions are formulatedgenerally by mixing P4 peptide at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, for example one that is non-toxicto recipients at the dosages and concentrations employed and iscompatible with other ingredients of the formulation. In addition, P4peptides (and/or opsonic antibodies, and/or antibiotic, and/orcomplement protein or fragments thereof) can be suspended in an aqueouscarrier, for example, in an isotonic buffer solution at a pH of about3.0 to about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to6.0, or 3.5 to about 5.0. Useful buffers include sodium citrate-citricacid and sodium phosphate-phosphoric acid, and sodium acetate/aceticacid buffers. The P4 peptide, optionally together with excipients,opsonic antibody, antibiotic and/or complement protein or fragmentsthereof, can also be in the form of a lyophilisate and can be made intoa solution prior to parenteral administration by the addition ofsuitable solvents. Solutions such as those that are used, for example,for parenteral administration can also be used as infusion solutions.

Pharmaceutical compositions can include an effective amount (such as atherapeutically effective amount) of P4 peptide, complement protein,antibiotic, and/or opsonic antibodies (for example, dissolved orsuspended) in a pharmaceutically acceptable carrier or excipient.Pharmaceutically acceptable carriers and/or pharmaceutically acceptableexcipients are known in the art and are described, for example, inRemington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975).

The nature of the carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch or magnesiumstearate. In addition, pharmaceutical compositions to be administeredcan contain minor amounts of non-toxic auxiliary substances, such aswetting or emulsifying agents, preservatives, and pH buffering agentsand the like, for example sodium acetate or sorbitan monolaurate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. For example, certainpharmaceutical compositions can include P4 peptide in water, mixed witha suitable surfactant, such as hydroxypropylcellulose. Dispersions alsocan be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Additional formulations are suitable for oral administration. Oralformulations can include excipients such as, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate and the like. The compositions(medicaments) typically take the form of solutions, suspensions,aerosols or powders. Exemplary formulations can be found in U.S. Patentpublication No. 20020031527. When the route is topical, the form may bea cream, ointment, salve or spray.

Typical subjects intended for treatment with the pharmaceuticalcompositions and methods of the present disclosure include humans, aswell as non-human primates and other animals. To identify subjects forprophylaxis or treatment according to the methods of the disclosure,accepted screening methods are employed to determine risk factorsassociated with a targeted or suspected disease or condition (forexample, an infection associated with a particular pathogen of interest)or to determine the status of an existing disease or condition in asubject. These screening methods include, for example, diagnosticmethods, such as various ELISA and other immunoassay methods, which areavailable and well known in the art to detect and/or characterizedisease-associated markers. These and other routine methods allow theclinician to select patients in need of therapy using the methods andpharmaceutical compositions of the disclosure.

An effective amount of the pharmaceutical composition is determinedbased on the intended goal, for example to inhibit and/or treat apathogenic infection of a human or non-human subject. The administrationof the pharmaceutical compositions of the disclosure can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, thepharmaceutical composition is provided in advance of any symptom. Theprophylactic administration of the compound serves to prevent orameliorate any subsequent disease process. When providedtherapeutically, the compound is provided at (or shortly after) theonset of a symptom of disease or infection.

For prophylactic and therapeutic purposes, the pharmaceuticalcompositions can be administered to the subject in a single bolusdelivery, via continuous delivery (for example, continuous transdermal,mucosal or intravenous delivery) over an extended time period, or in arepeated administration protocol (for example, by an hourly, daily orweekly, repeated administration protocol). The therapeutically effectivedosage of the compound can be provided as repeated doses within aprolonged prophylaxis or treatment regimen that will yield clinicallysignificant results to alleviate one or more symptoms or detectableconditions associated with a targeted disease or condition as set forthherein. Determination of effective dosages in this context is typicallybased on animal model studies followed up by human clinical trials andis guided by administration protocols that significantly reduce theoccurrence or severity of targeted disease symptoms or conditions in thesubject. Suitable models in this regard include, for example, murine,rat, porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (for example, immunologic andhistopathologic assays). Using such models, only ordinary calculationsand adjustments are required to determine an appropriate concentrationand dose to administer a therapeutically effective amount of the P4peptide (for example, amounts that are effective to alleviate one ormore symptoms of a targeted infection).

The appropriate dose will vary depending on the characteristics of thesubject, for example, whether the subject is a human or non-human, theage, weight, and other health considerations pertaining to the conditionor status of the subject, the mode, route of administration, and numberof doses, and whether the pharmaceutical composition includes both P4peptide alone or in conjunction with an opsonic antibody and/orantibiotic and/or complement protein, time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the therapeutic compositions for elicitingthe desired activity or biological response in the subject. Dosageregimens can be adjusted to provide an optimum prophylactic ortherapeutic response. A therapeutically effective amount is also one inwhich any toxic or detrimental side effects of the compound and/or otherbiologically active agent is outweighed in clinical terms bytherapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of a P4 peptide and/or otherbiologically active agent within the methods and formulations of thedisclosure is about 0.01 mg/kg body weight to about 10 mg/kg bodyweight, such as about 0.05 mg/kg to about 5 mg/kg body weight, or about0.2 mg/kg to about 2 mg/kg body weight.

Therapeutic compositions that include a disclosed therapeutic agent canbe delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudeket al., N. Engl. J. Med. 321:574, 1989) or by continuous subcutaneousinfusions, for example, using a mini-pump. An intravenous bag solutioncan also be employed. One factor in selecting an appropriate dose is theresult obtained, as measured by the methods disclosed here, as aredeemed appropriate by the practitioner. Other controlled release systemsare discussed in Langer (Science 249:1527-33, 1990).

In one example, a pump is implanted (for example see U.S. Pat. Nos.6,436,091; 5,939,380; and 5,993,414). Implantable drug infusion devicesare used to provide patients with a constant and long-term dosage orinfusion of a therapeutic agent. Such device can be categorized aseither active or passive.

Active drug or programmable infusion devices feature a pump or ametering system to deliver the agent into the patient's system. Anexample of such an active infusion device currently available is theMedtronic SYNCHROMED™ programmable pump. Passive infusion devices, incontrast, do not feature a pump, but rather rely upon a pressurized drugreservoir to deliver the agent of interest. An example of such a deviceincludes the Medtronic ISOMED™.

In particular examples, therapeutic compositions including a disclosedtherapeutic agent are administered by sustained-release systems.Suitable examples of sustained-release systems include suitablepolymeric materials (such as, semi-permeable polymer matrices in theform of shaped articles, for example films, or mirocapsules), suitablehydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, and sparingly soluble derivatives (such as, forexample, a sparingly soluble salt). Sustained-release compositions canbe administered orally, parenterally, intracistemally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), or as an oral or nasal spray. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman etal., Biopolymers 22:547-556, 1983, poly(2-hydroxyethyl methacrylate));(Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem.Tech. 12:98-105, 1982, ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (for example, U.S. Pat. No.5,055,303; U.S. Pat. No. 5,188,837; U.S. Pat. No. 4,235,871; U.S. Pat.No. 4,501,728; U.S. Pat. No. 4,837,028; U.S. Pat. No. 4,957,735; andU.S. Pat. No. 5,019,369; U.S. Pat. No. 5,055,303; U.S. Pat. No.5,514,670; U.S. Pat. No. 5,413,797; U.S. Pat. No. 5,268,164; U.S. Pat.No. 5,004,697; U.S. Pat. No. 4,902,505; U.S. Pat. No. 5,506,206; U.S.Pat. No. 5,271,961; U.S. Pat. No. 5,254,342; and U.S. Pat. No.5,534,496).

The pharmaceutical compositions (medicaments) can be prepared for use inprophylactic regimens and administered to human or non-human subjects toprotect against infection by a pathogen (or a plurality of pathogens).Thus, the pharmaceutical compositions typically contain apharmaceutically effective amount of P4 peptide and optionally apharmaceutically effective amount of opsonic antibody or a fragmentthereof, and/or antibiotic and/or complement protein or a fragmentthereof. In some cases the compositions are administered followinginfection, for example to treat the infection an increase pathogenclearance, in such applications, the pharmaceutical composition isadministered in a therapeutically effective amount. A therapeuticallyeffective amount is a quantity of a composition used to achieve adesired effect in a subject. For instance, this can be the amount of thecomposition necessary to inhibit infection by a pathogen, to increasepathogen clearance from the subject or to prevent or measurably alteroutward symptoms of pathogen infection from a subject. When administeredto a subject, a dosage will generally be used that will achieve targettissue concentrations that has been shown to achieve an in vitro or invivo effect.

C. Opsonic Antibodies

An immunogen, such as an immunogenic polysaccharide or an immunogenicpolypeptide (for example an immunogenic polysaccharide or an immunogenicpeptide derived from a pathogen, such as a viral, bacterial, or fungalpathogen, for example the viral, bacterial, or fungal pathogens listedabove) or a fragment or conservative variant thereof can be used toproduce opsonic antibodies which are immunoreactive or bind to anepitope on the surface of a pathogen, for example bind to particulateantigens and induce the opsonophagocytosis of the target pathogen byeffector cells. Polyclonal opsonic antibodies, antibodies which consistessentially of pooled opsonic monoclonal antibodies with differentepitopic specificities, as well as distinct monoclonal opsonic antibodypreparations are included.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al., “Production of PolyclonalAntisera,” in Immunochemical Protocols pages 1-5, Manson, ed., HumanaPress 1992; Coligan et al., “Production of Polyclonal Antisera inRabbits, Rats, Mice and Hamsters,” in: Current Protocols in Immunology,section 2.4.1, 1992.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature 256:495, 1975; Coligan et al.,sections 2.5.1-2.6.7; and Harlow et al., in: Antibodies: a LaboratoryManual, page 726, Cold Spring Harbor Pub., 1988. Briefly, monoclonalantibodies can be obtained by injecting mice with a compositioncomprising an antigen (for example an antigen derived from a pathogen),verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B lymphocytes, fusing the Blymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones that produce antibodies to theantigen, and isolating the antibodies from the hybridoma cultures.Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;Barnes et al., “Purification of Immunoglobulin G (IgG),” in: Methods inMolecular Biology, Vol. 10, pages 79-104, Humana Press, 1992.

Methods of in vitro and in vivo multiplication of monoclonal antibodiesare well known to those skilled in the art. Multiplication in vitro maybe carried out in suitable culture media such as Dulbecco's ModifiedEagle Medium or RPMI 1640 medium, optionally supplemented by a mammalianserum such as fetal calf serum or trace elements and growth-sustainingsupplements such as normal mouse peritoneal exudate cells, spleen cells,thymocytes or bone marrow macrophages. Production in vitro providesrelatively pure antibody preparations and allows scale-up to yield largeamounts of the desired antibodies. Large-scale hybridoma cultivation canbe carried out by homogenous suspension culture in an airlift reactor,in a continuous stirrer reactor, or in immobilized or entrapped cellculture. Multiplication in vivo may be carried out by injecting cellclones into mammals histocompatible with the parent cells, for example,syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. After oneto three weeks, the desired monoclonal antibody is recovered from thebody fluid of the animal.

Antibodies can also be derived from subhuman primate antibody. Generaltechniques for raising therapeutically useful antibodies in baboons canbe found, for example, in WO 91/11465, 1991, and Losman et al., Int. J.Cancer 46:310, 1990.

Alternatively, an antibody that specifically binds a polypeptide derivedfrom a pathogen can be derived from a humanized monoclonal antibody.Humanized monoclonal antibodies are produced by transferring mousecomplementarity determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and thensubstituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, forexample, by Orlandi et al., Proc. Nat'l Acad. Sci. U.S.A. 86:3833, 1989.Techniques for producing humanized monoclonal antibodies are described,for example, by Jones et al., Nature 321:522, 1986; Riechmann et al.,Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carteret al., Proc. Nat'l Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev.Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.

Antibodies can be derived from human antibody fragments isolated from acombinatorial immunoglobulin library. See, for example, Barbas et al.,in: Methods: a Companion to Methods in Enzymology, Vol. 2, page 119,1991; Winter et al., Ann. Rev. Immunol. 12:433, 1994. Cloning andexpression vectors that are useful for producing a human immunoglobulinphage library can be obtained, for example, from STRATAGENE® CloningSystems (La Jolla, Calif.).

In addition, antibodies can be derived from a human monoclonal antibody.Such antibodies are obtained from transgenic mice that have been“engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain loci are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994;and Taylor et al., Int. Immunol. 6:579, 1994.

Antibodies include intact molecules as well as fragments thereof, suchas Fab, F(ab′)₂, and Fv which are capable of binding the epitopicdeterminant. Methods of making these fragments are known in the art.(See for example, Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988). An epitope is any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent (Inbar et al., Proc. Nat'lAcad. Sci. U.S.A. 69:2659, 1972). Alternatively, the variable chains canbe linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. See, for example, Sandhu, supra.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing sFvsare known in the art (see Whitlow et al., Methods: a Companion toMethods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology11:1271, 1993; and Sandhu, supra).

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (Larrick et al., Methods: aCompanion to Methods in Enzymology, Vol. 2, page 106, 1991).

Antibodies can be prepared using an intact polypeptide, fragmentscontaining small peptides or polysaccharides of interest as theimmunizing antigen. A polypeptide or a peptide used to immunize ananimal can be derived from substantially purified polypeptide producedin host cells, in vitro translated cDNA, or chemical synthesis, whichcan be conjugated to a carrier protein, if desired. Such commonly usedcarriers which are chemically coupled to the peptide include keyholelimpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), andtetanus toxoid. The coupled peptide is then used to immunize the animal(for example, a mouse, a rat, or a rabbit).

Polyclonal or monoclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coliganet al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991).

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies, which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region that is the“image” of the epitope bound by the first monoclonal antibody.

Antibodies can be prepared by cloning techniques. Examples ofappropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises arefound in Sambrook et al., Molecular Cloning: A Laboratory Manual (2ndEd.), Vols. 1-3, Cold Spring Harbor Laboratory (1989), Berger and Kimmel(eds.), Guide to Molecular Cloning Techniques, Academic Press, Inc., SanDiego Calif. (1987), or Ausubel et al. (eds.), Current Protocols inMolecular Biology, Greene Publishing and Wiley-Interscience, NY (1987).Product information from manufacturers of biological reagents andexperimental equipment also provide useful information. Suchmanufacturers include the SIGMA chemical company (Saint Louis, Mo.), R&Dsystems (Minneapolis, Minn.), Pharmacia LKB Biotechnology (Piscataway,N.J.), CLONTECH® oratories, Inc. (Palo Alto, Calif.), Chem Genes Corp.,Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCOBRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),INVITROGEN™ (San Diego, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill

A substantially pure target antigen derived from a pathogen suitable foruse as an immunogen to produce opsonizing antibodies is isolated bypurification or recombinant expression (see section D). Concentration ofprotein in the final preparation is adjusted, for example, byconcentration on an Amicon filter device, to the level of a fewmicrograms per milliliter. Monoclonal or polyclonal antibody to theprotein can then be prepared as described by Harlow and Lane(Antibodies, A Laboratory Manual, Cold Spring Harbor Press. 1988).

Alternatively, antibodies may be raised against a synthetic peptidesynthesized on a commercially available peptide synthesizer based uponthe predicted amino acid or known sequence of the target orinternalizing receptor polypeptide (Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Press. 1988).

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3;Barnes et al., “Purification of Immunoglobulin G (IgG),” in: Methods inMolecular Biology, Vol. 10, pages 79-104, Humana Press, 1992.

Polyclonal or monoclonal antibodies can be further purified, forexample, by binding to and elution from a matrix to which thepolypeptide or a peptide to which the antibodies were raised is bound.Those of skill in the art will know of various techniques common in theimmunology arts for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies (See for example, Coliganet al., Unit 9, Current Protocols in Immunology, Wiley Interscience,1991).

To determine that a given antibody preparation (such as one produced ina mouse) specifically binds the target or internalizing receptorpolypeptide of interest by Western blotting, total cellular proteincontaining the target or internalizing receptor polypeptide is extractedfrom murine myeloma cells and electrophoresed on a SDS-polyacrylamidegel. The proteins are then transferred to a membrane (for example,nitrocellulose), and the test antibody preparation is incubated with themembrane. After washing the membrane to remove non-specifically boundantibodies, the presence of specifically bound antibodies is detected bythe use of an anti-mouse antibody conjugated to an enzyme such asalkaline phosphatase; application of the substrate5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium results inthe production of a dense blue compound by immuno-localized alkalinephosphatase. Antibodies which specifically bind a target orinternalizing receptor polypeptide of interest will, by this technique,be shown to bind to the target or internalizing receptor polypeptideband (which will be localized at a given position on the gel determinedby its molecular weight). Non-specific binding of the antibody to otherproteins (such as serum albumin) may occur and may be detectable as aweak signal on the Western blot. The non-specific nature of this bindingwill be recognized by one skilled in the art by the weak signal and/orunrelated portion obtained on the Western blot relative to the strongprimary signal arising from the specific antibody-target orinternalizing receptor polypeptide binding.

D. Peptide Production

The P4 peptides, complement peptides, and peptides derived frompathogens can be prepared by cloning techniques. Examples of appropriatecloning and sequencing techniques, and instructions sufficient to directpersons of skill through many cloning exercises are found in Sambrook etal., Molecular Cloning: A Laboratory Manual (2nd Ed.), Vols. 1-3, ColdSpring Harbor Laboratory (1989), Berger and Kimmel (eds.), Guide toMolecular Cloning Techniques, Academic Press, Inc., San Diego Calif.(1987), or Ausubel et al. (eds.), Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience, NY (1987). Productinformation from manufacturers of biological reagents and experimentalequipment also provide useful information. Such manufacturers includethe SIGMA chemical company (Saint Louis, Mo.), R&D systems (Minneapolis,Minn.), Pharmacia LKB Biotechnology (Piscataway, N.J.), CLONTECH®laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp., AldrichChemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL LifeTechnologies, Inc. (Gaithersburg, Md.), Fluka Chemica-BiochemikaAnalytika (Fluka Chemie AG, Buchs, Switzerland), INVITROGEN™ (San Diego,Calif.), and Applied Biosystems (Foster City, Calif.), as well as manyother commercial sources known to one of skill In some examples,peptides, such as complement peptides are purified from a subject, forexample from a blood fraction of a subject, such as serum obtained froma subject.

In some embodiments, the peptides are produced recombinantly, forexample from cells transformed or transfected with polynucleotidesencoding the peptides or portion thereof. Methods for the manipulationand insertion of the nucleic acids encoding the peptides of thisdisclosure or portions thereof into vectors for the expression ofpolypeptides are well known in the art (see for example, Sambrook etal., Molecular Cloning, a Laboratory Manual, 2d edition, Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,Current Protocols in Molecular Biology, Greene Publishing Associates andJohn Wiley & Sons, New York, N.Y., 1994).

The nucleic acid constructs encoding the peptides or portions thereof ofthis disclosure can be inserted into plasmids. However, other vectors(for example, viral vectors, phage, cosmids, etc.) can be utilized toreplicate the nucleic acids. In the context of this disclosure, thenucleic acid constructs typically are expression vectors that contain apromoter sequence which facilitates the efficient transcription of theinserted genetic sequence of the host. The expression vector typicallycontains an origin of replication, a promoter, as well as specificnucleic acid sequences that allow phenotypic selection of thetransformed cells.

More generally, polynucleotide sequences encoding peptides or portionsthereof of this disclosure can be operably linked to any promoter and/orenhancer that is capable of driving expression of the nucleic acidfollowing introduction into a host cell. A promoter is an array ofnucleic acid control sequences that directs transcription of a nucleicacid. Both constitutive and inducible promoters are included (see, forexample, Bitter et al., Methods in Enzymology 153:516-544, 1987).

DNA sequences encoding peptides or portions thereof can be expressed invitro by DNA transfer into a suitable host cell. The cell may beprokaryotic or eukaryotic. Hosts can include microbial, yeast, insect,and mammalian organisms. The term also includes any progeny of thesubject host cell. Methods of stable transfer, meaning that the foreignDNA is continuously maintained in the host, are known in the art.

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques that are well known to those of ordinary skillin the art. Where the host is prokaryotic, such as E. coli, competentcells which are capable of DNA uptake can be prepared from cellsharvested after exponential growth phase and subsequently treated by theCaCl₂ method using procedures well known in the art. Alternatively,MgCl₂ or RbCl can be used. Transformation can also be performed afterforming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with polynucleotide sequences encoding peptides orportions thereof, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).Peptides can then be purified for host cells using methods known in theart.

Immunogenic peptides derived from pathogens and/or P4 peptides also maybe produced, for example by chemical synthesis by any of a number ofmanual or automated methods of synthesis known in the art. For example,solid phase peptide synthesis (SPPS) is carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethylpolystyrene (HMP) or Sasrin resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides.

Fmoc-derivatized amino acids are prepared from the appropriate precursoramino acids by tritylation and triphenylmethanol in trifluoroaceticacid, followed by Fmoc derivitization as described by Atherton et al.Solid Phase Peptide Synthesis, IRL Press: Oxford, 1989.

Sasrin resin-bound peptides are cleaved using a solution of 1% TFA indichloromethane to yield the protected peptide. Where appropriate,protected peptide precursors are cyclized between the amino- andcarboxyl-termini by reaction of the amino-terminal free amine andcarboxyl-terminal free acid using diphenylphosphorylazide in nascentpeptides wherein the amino acid sidechains are protected.

HMP or Rink amide resin-bound products are routinely cleaved andprotected sidechain-containing cyclized peptides deprotected using asolution comprised of trifluoroacetic acid (TFA), optionally alsocomprising water, thioanisole, and ethanedithiol, in ratios of100:5:5:2.5, for 0.5-3 hours at room temperature.

Crude peptides are purified by preparative high pressure liquidchromatography (HPLC), for example using a Waters Delta-Pak C18 columnand gradient elution with 0.1% TFA in water modified with acetonitrile.After column elution, acetonitrile is evaporated from the elutedfractions, which are then lyophilized. The identity of each product soproduced and purified may be confirmed by fast atom bombardment massspectroscopy (FABMS) or electrospray mass spectroscopy (ESMS).

Peptides produced by such methods can be used to produce opsonicantibodies for the pathogens from which the immunogenic peptides arederived. Methods of determining the opsonic ability of an antibody canbe determined by methods, known to one of skill in the art, for examplethe methods described in International Patent Application No.PCT/US2006/015499 and U.S. patent application Ser. No. 11/910,517 whichare incorporated herein by reference in their entirety.

The following examples are provided to illustrate particular features ofcertain embodiments. However, the particular features described belowshould not be construed as limitations on the scope of the disclosure,but rather as examples from which equivalents will be recognized bythose of ordinary skill in the art.

EXAMPLES Example 1

This example describes tests demonstrating enhancement ofopsonophagocytosis by the P4 peptide.

Materials and Methods

Peptide Synthesis.

The amino acid sequences of the peptides designated P4, P6, and P7 havebeen described previously (see e.g. Rajam et al., Microb Pathog 2008;44:186-96; Romero-Steiner et al., Vaccine 2006; 24:3224-31). The purepeptides with a free N- and C-terminus were synthesized and lyophilizedat the Centers for Disease Control and Prevention (CDC) and the EmoryUniversity Microchemical Facility. Peptides used in this Example weresynthesized in an Advanced ChemTech 396 multiple peptide synthesizer bymeans of standard and modified 9-fluorenylmethoxycarbonyl protocols.Lyophilized peptide was resuspended in diethylpyrocarbonate (DEPC)water, sonicated for 3 minutes for dissolution, and stored at −70° C.Two peptides, P6 and/or P7, were derived from the P4 sequence. Thesepeptides had no activation effect on the eukaryotic cells tested (seeRajam et al., Microb Pathog 44:186-96, 2008). These peptides were usedas negative controls in all in vitro tests described in this Example.

Species-Specific Antibodies Used in this Example.

Globulin (Gamunex®) has been used as a source of pneumococcalserotype-specific polysaccharide (Ps) antibodies (see e.g. Rajam et al.,Clin Vaccine Immunol, 14:1223-7, 2007; Romero-Steiner et al., Clin DiagnLab Immunol 10:1019-24, 2003). QC2, QC5, and QC268 are CDC in-housequality-control human sera with titers assigned to target organisms (seee.g. Martinez et al., Clin Diagn Lab Immunol 9:485-8, 2002; Martinez etal., Clin Vaccine Immunol 13:459-66, 2006]. A monoclonal antibody withspecificity for pneumococcal surface adhesin A as described bySrivastava et al. (Hybridoma 2000; 19:23-31), 8G12G11B10 (8G12), wasalso used as a source of anti-streptococcal protein antibody. These serawere selected to ensure the presence of specific antibodies anddemonstrate the specificity of P4-mediated immune enhancement. The invitro trial design involved direct comparison of changes inopsonophagocytic killing (OPK) or uptake in the presence or absence ofP4.

OPK Assay.

In this Example, the reference OPK assay, as described in Romero-Steineret al., Clin Diagn Lab Immunol, 4:415-22, 1997, was used with humanpromyelocytic leukemia cells (HL-60) differentiated into granulocytes.Gamma globulin was used as the source for serotype-specific antibodiesfor S. pneumoniae serotype 3 (WU2) was propagated, stored, and used inthis assay as described by Romero-Steiner et al., Clin Vaccine Immunol,13:165-9, 2006 and Rajam et al., Clin Vaccine Immunol, 14:1223-7, 2007.P4 peptide solution (100 μg/mL) was added to the OPK assay mixture atthe preopsonization stage, and the control wells received 10 μL of DEPCwater instead. P4-mediated enhancement of OPK was also assessed with S.pneumoniae serotypes 6B, 15B, 15C, and 19A, using gamma globulin or8G12.

Flow Cytometric Opsonophagocytic Uptake Assay.

The flow cytometric opsonophagocytic assay (fOPA) was performed withHL-60 cells differentiated into granulocytes or monocytes as describedby Martinez et al., Clin Diagn Lab Immunol 9:485-8, 2002; Martinez etal., Clin Vaccine Immunol 13:459-66, 2006; and Mezzatesta et al., InfectImmun 42:99-105, 1983. In-house quality-control sera (QC5 and QC268)were used as a source for serotypespecific antibodies against thecapsular Ps of S. pneumoniae serotype 14 and Neisseria meningitides A,respectively. Polystyrene beads were covalently linked to S. pneumoniaeand non-S. pneumoniae antigens, as described by Martinez et al., ClinDiagn Lab Immunol 9:485-8, 2002; and Martinez et al., Clin VaccineImmunol 13:459-66, 2006 and used in fOPA. P4 peptide solution (100μg/mL) was added to the fOPA mixture at the preopsonization stage, andthe control wells received 10 μL of DEPC water instead.

OXYBURST® Labeling of S. pneumoniae Isolate.

To demonstrate the enhancement of the intracellular respiratory burst inthe effector cells in response to P4-mediated activation, anOXYBURST®-labeled S. pneumoniae isolate was used. A loopful of thefrozen stock of S. pneumoniae serotype 23F was grown overnight (37° C.in 5% CO₂) in Todd-Hewitt broth (Difco) supplemented with 0.5% yeastextract (THYE). A loopful of the overnight culture was transferred to 1mL of fresh THYE broth and incubated for 3 hours. From this, 200 μL wastransferred to 5 mL of THYE broth and incubated for 3 hours, after which1 mL was transferred to 5 mL of THYE broth and incubated for another 3hours (all incubations were done at 37° C. in 5% CO₂). After the thirdpassage, the bacterial suspension was centrifuged at 6000 g for 10minutes and resuspended in 1 mL of 0.01 mol/L phosphate buffered saline(PBS). OXYBURST® stain (INVITROGEN®) was reconstituted with 1 mL ofdeionized water, and 50 μL was added to the 1-mL bacterial suspension.This was allowed to mix thoroughly in a rotary shaker overnight at 4° C.After that, the OXYBURST®-labeled bacterial suspension was washed twicein PBS and used as a source of antigen instead of polystyrene beads infOPA. In-house quality-control serum (QC2) was used in this assay.

Isolation of Polymorphonuclear Leukocytes from Human Blood.

Heparinized venous blood was obtained from the Emory Blood DonorServices. A leukocyte separation kit, HISTOPAQUE®-1119 (Sigma), was usedto separate granulocytes from the blood, in accordance with the methodrecommended by the manufacturer.

Mouse strains. Mice (Mus musculus) of strain Swiss Webster (ND4-SW) wereobtained from Charles River Laboratories. Mice used in this study were6-10 weeks old. All experiments were approved by the institutionalcommittee and were conducted according to institutional ethicalguidelines for animal experiments and safety guidelines.

Bacterial Strains.

S. pneumoniae WU2 (serotype 3) was used for mouse infections. This S.pneumoniae isolate was selected from the Streptococcal ReferenceLaboratory, CDC. Briefly, S. pneumoniae isolate (frozen stock) wasstreaked on a blood agar plate (blood agar base plus 5% sheep blood) andincubated (at 37° C. in 5% CO²) for 18-24 hours. S. pneumoniae colonieson blood agar plates were scraped with an inoculation loop and grown in5 mL of THYE broth for ˜4 hours (at 37° C. in 5% CO²) until midlog phase(optical density read at 492 nm, 0.5-0.6). This culture (1.5 mL) wascentrifuged in a 2-mL polypropylene screw-cap tube at 10,000 g for 5minutes, and the wet pellet was resuspended in 1 mL of 0.1 mol/L PBS (pH7.2). The 1-mL bacterial suspension was placed on ice and used forinfections. It was also diluted 10⁻⁶ with PBS, and the viable bacterialload was enumerated on blood agar plates. The average viable bacterialload was 4×10⁷ cells/mL.

Intranasal Infection.

Mice were intranasally infected with S. pneumoniae by means of methodsdescribed by Briles et al., J Infect Dis 188:339-48, 2003. Briefly, amouse was injected intraperitoneally with 20 μL of KETASET® (100 mg/mLketamine hydrochloride; Wyeth). Once the mouse was lethargic, 40 μL ofthe previously prepared bacterial suspension was dispensed drop by dropclose to the nose, allowing the mouse to inhale the infection.

Intraperitoneal (ip) and Intravenous (iv) Therapy.

Infection with 4×10⁷ cells of S. pneumoniae WU2 per mouse resulted inmoribund characteristics at 48 hours in 50%-60% of animals. At 72 hours,all the infected mice were moribund (n=60; moribund score, 2-3 [seebelow]). At 72 and 96 hours after infection, 40 animals were passivelyimmunized with gamma globulin (100 μL/mouse; iv, n=20; ip, n=20). Aftera time lapse of 20 minutes, allowing for possible preopsonization invivo, 20 of the passively immunized mice (intravenous (iv), n=10;intraperitoneal (ip), n=10) received P4 (100 μg; 100 μL/mouse) throughan iv or ip route. Control mice were given DEPC water (100 μL) or P4alone. Initially, P4 peptide was tested for toxicity in mice at 1, 10,100, and 1000 μg. P4 was injected ip into 10-week-old ND4-SW mice at aconstant volume of 100 μL. P4 had no apparent toxic effect on mice, evenat doses of 1000 μg/mouse.

Scoring of Moribund Characteristics.

Mice were monitored and visually scored twice daily for moribundcharacteristics. Mice were ranked on a scale of 5 to 0, in which 5indicated healthy with normal coat, skin, eyes, breathing, andactivity/movement; 4, healthy but beginning to look sick, ruffled coat;3, sick, ruffled coat, decreased activity; 2, very sick, ruffled coat,decreased activity, eye secretions; 1, near death, ruffled coat, littleor no activity, eye secretions, decreased breathing (these animals were,hence, euthanized); and 0, dead.

Cytokine Analysis.

P4-treated and control mice were killed and decapitated. The blood wasquickly collected in cryovials from the base of the neck and allowed tostand at 4° C. for 30 minutes. The tube was then centrifuged at 1000 gfor 10 minutes. Serum samples were collected and used immediately forcytokine analysis. Cytokines were analyzed in mouse serum by means ofthe LUMINEX®-based LINCOPLEX™ mouse 22-plex cytokine kit(MCYTO-70K-PMX22; LINCO Research), using the manufacturer-recommendedprotocol.

Statistics.

All in vitro experiments were performed in triplicate on 3 separateassay days, unless specified otherwise. The in vivo challengeexperiments were repeated >5 times. The number of moribund animals aftertreatment was recorded for 166 hours, and the data were analyzed forsignificant differences among various groups by use of at test withpaired samples for means (Microsoft® Excel 2003).

Results

OPK Assay.

P4 peptide was tested for its potential to enhance opsonophagocytosis invitro, and the data are given in FIG. 1. Data presented in FIG. 1A showthat the P4-mediated increase in OPK of S. pneumoniae serotype 3 (WU2)was dependent on the antibody concentration. Although a 35% increase inOPK is seen at a 1:8 dilution of gamma globulin, the effect titrates outwith the dilution of the antiserum (FIG. 1A). The P4-mediated increasein OPK was complement dependent, because no increase in OPK with P4 overcontrol was observed in the absence of a complement source (FIG. 1B).The P4-mediated enhancement of OPK was dependent on the concentration ofP4 in the reaction mixture. Although no change in OPK was seen with P4supplementation at 5 μg/mL, a gradual increase was seen with an increasein P4 concentration (for 10 μg/mL, 8%; for 50 μg/mL, 30%; for 100 μg/mL,35%). At a concentration of 100 μg/mL, the P4-mediated increase in OPKplateaued (FIG. 1C). A similar P4-mediated increase in OPK was alsorecorded with S. pneumoniae serotypes 6B, 15B, 15C, and 19A, using gammaglobulin or 8G12 as the source of specific antibodies.

Opsonophagocytic Uptake (OPU) Assay.

P4-mediated enhancement of opsonophagocytosis was tested for changes inthe intraphagocytic respiratory burst by means of OXYBURST® labeled S.pneumoniae serotype 23F (DS3848-03). P4-mediated increase in OPU wascharacterized by an increase in the intraphagocytic respiratory burstthat titrated out with the antibody dilution (FIG. 2A). Data presentedin FIGS. 2B and 2C show P4 as being pluripotent in activating differenteffector cells and enhancing OPU in the presence of antigen-specificantibodies and complement. There was a 50% increase in OPU of S.pneumoniae serotype 14 Ps beads by the granulocytes isolated from freshhuman blood, which titrated out with the antibody dilution (for 1:64,52%; for 1:128, 48%; for 1:256, 25%; for 1:512, 5%) (FIG. 2B). FIG. 2Cshows that P4 can enhance in vitro opsonophagocytosis of non-S.pneumoniae antigens in the presence of specific antibodies and effectorcells. A P4-mediated increase in the OPU of beads coated with N.meningitidis APs was recorded, with HL-60 cells differentiated intomonocytes (FIG. 2C).

In Vivo Studies.

Mice infected with S. pneumoniae WU2 were passively immunized with gammaglobulin and/or P4 at 72 and 96 hours after infection. Although theuntreated mice had 10% survival (1 in 10), mice treated with gammaglobulin alone (both iv and ip) had 30% survival. On the other hand, 8(80%) of 10 mice (P<0.001) treated with iv and 6 (60%) of 10 mice(P<0.001) treated with ip gamma globulin and P4 exhibited completeremission of bacteremia and moribundity (FIG. 3). Cytokine analysis ofmouse serum samples showed no consistent pattern or changes in cytokinelevels in the rescued animals.

Example 2

This example describes the evaluation of co-administration of P4 peptideand antibiotics as a novel therapeutic approach to treating severepneumococcal infection.

Materials and Methods

Bacterium, Peptide, Antibodies, and Antibiotic Used in this Example.

S. pneumoniae serotype 3 (WU2) was used for mouse infections asdescribed previously (Rajam et al. J. Infect. Dis. 199:1233-1238, 1999).P4, a 28-amino-acid peptide, was synthesized, purified, and prepared forcombination therapy as described previously (Carlone et al. Microb.Pathog. 44:186-196, 2008). Gamma globulin (intravenous immunoglobulin[IVIG]; Gamunex, Telecris, N.C.) was used as a source of Pncserotype-specific polysaccharide antibodies (Frasch and Scott Clin.Diagn. Lab. Immunol. 11:1158-1164, 2004; Rajam et al., Clin. VaccineImmunol. 14:1223-1227, 2007; Romero-Steiner et al., Clin. Diagn. Lab.Immunol. 10:1019-1024, 2003). Ceftriaxone (catalog no. C5793;Sigma-Aldrich, St. Louis, Mo.), an expanded-spectrum cephalosporin, wasdissolved in phosphate-buffered saline (0.01 M), and working dilutionsin phosphate-buffered saline were made for mouse inoculations.

Mice Used in this Example.

Female Swiss Webster mice (Charles River Laboratories, Wilmington,Mass.) 6 to 10 weeks of age were used in this study. All experimentswere approved by the Institutional Animal Care and Use Committee (IACUC)and conducted according to the institutional ethical guidelines foranimal experiments and safety guidelines.

Intranasal Infection.

Intranasal infections of mice with a Pnc isolate were carried out byadopting the methodology described by Briles et al., 2003. Briefly, amouse was injected intraperitoneally (ip) with 20 μl of 100-mg/mlketamine hydrochloride (KETASET®; Wyeth). Once the mouse was lethargic,40 μl of the bacterial suspension (˜2.1×10⁷ cells/mouse) was dispenseddrop by drop close to the nose, allowing the mouse to inhale thebacteria. Scruffiness combined with a hunched posture or lethargyindicated moribundity in a mouse. Most mice (80%) were moribund at 48hours postexposure. Moribund mice were divided into various control andtreatment groups (n=10/group) as shown in Table 1. Control groupsincluded untreated animals or animals that received P4 alone, IVIGalone, or ceftriaxone (at 0.3, 3.0, 300, or 3,000 μg/mouse) alone.Treatment groups included mice that received a single inoculumcontaining P4 and IVIG with or without ceftriaxone at one of threedifferent doses (0.3, 3.0, or 300 μg/mouse).

Combination Therapy.

Mice were restrained using a Tailveiner restrainer (model no. TV-150;Braintree Scientific, Braintree, Mass.). IVIG and/or P4 was administeredintravenously (iv) using a 25-gauge needle and a 1-ml syringe. IVIG(100-μl volume/mouse) was administered first, followed 20 min later byP4 (50 μg in a 100-μl volume/mouse). Ceftriaxone was administered (ip ina 100-μl volume/mouse) 30 min after P4 administration. Animals weremonitored daily for 7 days or (in the repeat infection study) 36 dayspost treatment for clinical signs of disease progression. For repeattherapy, mice rescued with P4 combination therapy were reinfected on day28 post treatment and treated again with P4 combination therapy. Eventhough 25 to 30% of mice in the control group survived the firstchallenge, they later either succumbed to infection or were terminallyill and hence were humanely euthanized. Hence, there were no controlmice from the first challenge for the repeat therapy.

Test for Bacteremia.

Blood samples from P4-treated and untreated mice were collected (Rajamet al., J. Infect. Dis. 199:1233-1238, 2009), and 100-0 aliquots of theheparinized blood samples were spread onto plates of blood agar (bloodagar base plus 5% sheep blood plus gentamicin [2.5 mg/liter]). Theplates were incubated for 18 to 24 hours at 37° C. in 5% CO², andbacteria were counted.

P4-Enhanced Opsonophagocytosis.

We used the in vitro opsonophagocytic killing assay (OPKA) as describedpreviously by Romero-Steiner et al. (Romero-Steiner et al., Clin. Diagn.Lab. Immunol. 4:415-422, 1997) with polymorphonuclear leukocytes (PMNs)isolated from mice (Devi et al., Indian J. Physiol. Pharmacol.39:354-360, 1995). Peripheral blood samples were collected from mice 1and 2 hours postinfection (with Pnc WU2) or from uninfected controls asdescribed previously (Frasch and Scott, 2004), and the buffy coatfractions were separated (Devi et al, 1995) and used as the source ofeffector cells. Gamma globulin (Gamunex, Telecris, N.C.) was used as theserotype-specific antibody source. S. pneumoniae serotype 3 (WU2) waspropagated, stored, and used in this assay as described previously(Rajam et al., 2007; Romero-Steiner, 2006). A 100-μg/ml P4 peptidesolution was added to the OPKA mixture at the preopsonization stage, andthe control wells received diethyl pyrocarbonate water.

ELISA for Anti-P4 IgG.

Blood samples from mice treated with P4 therapy were collected 14 dayspost infection, and the serum fractions were separated by adopting themethodology described previously (Rajam, 2009). An enzyme-linkedimmunosorbent assay (ELISA) was performed to detect and quantifyantiprotein immunoglobulin G (IgG) as described previously, with minormodifications (Scott, et al., Clin. Diagn. Lab. Immunol. 12:1195-1201,2005; Scott et al., J. Infect. Dis. 186:220-226, 2002). Briefly, ELISAplates were coated with the P4 peptide at a 5-μg/ml concentration.Plates were incubated at 4° C. overnight and used to detect and quantifyanti-P4 IgG in mouse sera. Horseradish peroxidase-labeled anti-mouse IgG(Sigma, St. Louis, Mo.) was used as the reporter antibody. SUREBLUE®3,3′,5,5′-tetramethylbenzidine (KPL, Gaithersburg, Md.) was used as thesubstrate, and 1 N HCl was used as the stop solution. Samples wereassayed in triplicate, and suitable positive and negative controls wereincluded.

Statistics.

The in vivo combination therapy experiments were repeated three to fivetimes, and repeat therapy was performed once. The numbers of moribundanimals up to 7 and 36 days after the combination and repeat therapies,respectively, were recorded, and the data were analyzed for significantdifferences among various groups by using the paired two-sample t testfor means in MICROSOFT® Excel 2003.

Results

Combination Therapy.

Untreated control mice (n=10) had a 30% survival rate at 168 hours. Micetreated with ceftriaxone alone at 3,000 μg had a 90% survival rate (FIG.4). On the other hand, ceftriaxone at lower doses (300, 3.0, and 0.3 μg)or P4 alone offered poor protection, with survival rates comparable tothat of untreated controls. Treating moribund mice with a P4concentration of 50 μg with IVIG administered in a single dose led to asurvival rate of 70% (FIG. 4). Combining a low dose (300 μg) ofceftriaxone with this IVIG-P4 therapeutic mixture increased the mousesurvival rate to 100%, significantly better than that of untreatedcontrols (P<0.05) (FIG. 4).

Repeat Therapy.

Mice (n=10) rescued from fatal S. pneumoniae WU2 infection withcombination (P4, WIG, and ceftriaxone) therapy were reinfected with S.pneumoniae WU2 after 28 days and retreated with the combination therapywhen they appeared to be moribund. P4-mediated combination therapyrescued all the infected animals (FIG. 5).

Test for Bacteremia.

Blood samples from P4-treated and untreated mice were drawn and testedfor bacteremia. Blood samples from the untreated control mice containedloads of bacteria too numerous to count. Samples from treated animalshad no bacteremia.

P4-Enhanced Opsonophagocytosis.

An OPKA with PMNs from either infected or uninfected mice was performed.The addition of P4 significantly increased (by >80%; P<0.05) the invitro opsonophagocytic killing of Pnc (WU2) over the control level inthe presence of serotype-specific IgG (FIG. 6).

ELISA for Anti-P4 IgG.

Serum samples from P4-treated mice were tested for anti-P4 by using amouse IgG-specific ELISA. All the samples were negative for anti-P4 IgG.

TABLE 1 Study design Use^(b) of: Ceftriaxone Gamma 3,000 TreatmentGroup^(a) P4 globulin μg 300 μg 3.0 μg 0.3 μg Combination 1 − − − − − −therapy 2 + − − − − − 3 − + − − − − 4 − − + − − − 5 − − − + − − 6 − − −− + − 7 − − − − − + 8 + + − − − − 9 + + − + − − 10 + + − − + − 11 + + −− − + Repeat 10 + + − − + − therapy ^(a)n = 10/group. All groupsreceived S. pneumoniae serotype 3 (WU2) intranasally at ~2.1 × 10⁷cells/40 μl/mouse. ^(b)P4 at 50 μg in a 100-μl volume and 100 μl ofgamma globulin were administered iv; ceftriaxone was administered ip atthe indicated doses in a 100-μl volume. +, included in the therapeuticmixture; −, not included in the therapeutic mixture.

Example 3 Treatment of Subjects with P4 Peptide

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from a pathogen of interest(such as the pathogens listed in the summary of terms) that can betreated by opsonophagocytosis of the pathogen of interest byadministration of one or more of P4 peptides, and optionally an opsonicantibody and/or complement protein. In some examples, the one or more P4peptides is administered without an opsonic antibody and or and/orcomplement protein. In particular examples, the method includesscreening a subject having, thought to have, or at risk of having (forexample due to impaired immunity, physiological status, or exposure to apathogen) a pathogenic infection. Subjects of an unknown infectionstatus can be examined to determine if they have an infection, forexample using serological tests, physical examination, enzyme-linkedimmunosorbent assay (ELISA), radiological screening or other diagnostictechnique known to those of skill in the art. In some examples, asubject is selected that has a pathogenic infection or is at risk ofacquiring a pathogen infection from a pathogen that does not expresspneumococcal surface adhesin A (PsaA), for example the subject does nothave a Streptococcus pneumoniae infection. Subjects found to (or knownto) have a pathogenic infection and thereby treatable by administrationof P4 peptide are selected to receive P4 peptide. Subjects may also beselected who are at risk of developing a pathogenic infection forexample, the elderly, the immunocompromised and the very young, such asinfants.

Subjects selected for treatment can be administered a therapeutic amountof P4 peptide. The P4 peptide can be administered at doses of 1 μg/kgbody weight to about 1 mg/kg body weight per dose, such as 1 μg/kg bodyweight −100 μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kgbody weight per dose, or 500 μg/kg body weight—1000 μg/kg body weightper dose or even greater. However, the particular dose can be determinedby a skilled clinician. The agent can be administered in several doses,for example continuously, daily, weekly, or monthly.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

Example 4 Treatment of Subjects with P4 Peptide and Opsonic Antibodies

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from a pathogen of interest(such as the pathogens listed in the summary of terms) that can betreated by opsonophagocytosis of the pathogen of interest byadministration of one or more of P4 peptides and an opsonic antibodythat specifically binds an antigen present on the surface of thepathogen of interest. In particular examples, the method includesscreening a subject having, thought to have or at risk of having apathogenic infection. Subjects of an unknown infection status can beexamined to determine if they have an infection, for example usingserological tests, physical examination, enzyme-linked immunosorbentassay (ELISA), radiological screening or other diagnostic techniqueknown to those of skill in the art. In some examples, subjects arescreened to identify a particular pathogen of interest, with aserological test, or with a nucleic acid probe specific for a pathogenof interest, or even a panel of nucleic acid probes, such as an array,that can identify several pathogens simultaneously. Subjects found to(or known to) have a pathogenic infection from a pathogen of interest,for example Streptococcus pneumoniae, Streptococcus pyogenes, Neisseriameningitides or Staphylococcus aureus, such as methicillin resistantStaphylococcus aureus (MRSA), and thereby treatable by administration ofP4 peptide in conjunction with a opsonic antibody specific for thedetected pathogen of interest are selected for administration of P4peptide and the opsonic antibody specific for the pathogen of interest.Subjects may also be selected who are at risk of developing a pathogenicinfection for example, subjects exposed to a known pathogen of interest,the elderly, the immunocompromised and the very young, such as infants.

Subjects selected for treatment can be administered a therapeutic amountof P4 peptide. The P4 peptide can be administered at doses of 1 μg/kgbody weight to about 1 mg/kg body weight per dose, such as 1 μg/kg bodyweight −100 μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kgbody weight per dose, or 500 μg/kg body weight −1000 μg/kg body weightper dose. Subjects are administered a therapeutic amount of opsonicantibody that is specific for the identified pathogen of interest. Theopsonic antibody can be administered at doses of 1 μg/kg body weight toabout 1 mg/kg body weight per dose, such as 1 μg/kg body weight −100μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kg body weightper dose, or 500 μg/kg body weight −1000 μg/kg body weight per dose.However, the particular dose can be determined by a skilled clinician.The P4 peptide can be administered concurrently or sequentially withopsonic antibody. The P4 peptide and/or the opsonic antibody can beadministered in one or several doses, for example continuously, daily,weekly, or monthly. When administered sequentially the time separatingthe administration of the P4 peptide and opsonic antibody can beseconds, minutes, hours, days, or even weeks.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

Example 5 Treatment of Subjects with a Combination of Antibiotics, P4Peptide and Opsonic Antibodies

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from a pathogen of interest(such as the pathogens listed in the summary of terms) that can betreated by opsonophagocytosis of the pathogen of interest byadministration of one or more of P4 peptides and an opsonic antibodythat specifically binds an antigen present on the surface of thepathogen of interest. In particular examples, the method includesscreening a subject having, thought to have or at risk of having apathogenic infection. Subjects of an unknown infection status can beexamined to determine if they have an infection, for example usingserological tests, physical examination, enzyme-linked immunosorbentassay (ELISA), radiological screening or other diagnostic techniqueknown to those of skill in the art. In some examples, subjects arescreened to identify a particular pathogen of interest, with aserological test, or with a nucleic acid probe specific for a pathogenof interest, or even a panel of nucleic acid probes, such as an array,that can identify several pathogens simultaneously. Subjects found to(or known to) have a pathogenic infection from a pathogen of interest,for example Streptococcus pneumoniae, Streptococcus pyogenes, Neisseriameningitides or Staphylococcus aureus, such as methicillin resistantStaphylococcus aureus (MRSA), and thereby treatable by administration ofP4 peptide in conjunction with antibiotics and a opsonic antibodyspecific for the detected pathogen of interest are selected foradministration of P4 peptide the antibiotic and the opsonic antibodyspecific for the pathogen of interest. Subjects may also be selected whoare at risk of developing a pathogenic infection for example, subjectsexposed to a known pathogen of interest, the elderly, theimmunocompromised and the very young, such as infants.

Subjects selected for treatment are administered a therapeutic amount ofP4 peptide. The P4 peptide can be administered at doses of 1 μg/kg bodyweight to about 1 mg/kg body weight per dose, such as 1 μg/kg bodyweight −100 μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kgbody weight per dose, or 500 μg/kg body weight −1000 μg/kg body weightper dose. Subjects are administered a therapeutic amount of opsonicantibody that is specific for the identified pathogen of interest. Theopsonic antibody can be administered at doses of 1 μg/kg body weight toabout 1 mg/kg body weight per dose, such as 1 μg/kg body weight −100μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kg body weightper dose, or 500 μg/kg body weight −1000 μg/kg body weight per dose.However, the particular dose can be determined by a skilled clinician.Subjects selected for treatment are administered a therapeutic amount ofP4 peptide. The antibiotic can be administered at doses of 1 μg/kg bodyweight to about 1 mg/kg body weight per dose( ) depending upon suchfactors as the strength and/or type of antibiotic), such as 1 μg/kg bodyweight −100 μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kgbody weight per dose, or 500 μg/kg body weight −1000 μg/kg body weightper dose. The P4 peptide can be administered concurrently orsequentially with the antibiotic and opsonic antibody.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

Example 6 Treatment of Subjects with P4 Peptide and Antibiotic

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from a pathogen of interest(such as the pathogens listed in the summary of terms) that can betreated by opsonophagocytosis of the pathogen of interest byadministration of one or more of P4 peptides and an opsonic antibodythat specifically binds an antigen present on the surface of thepathogen of interest. In particular examples, the method includesscreening a subject having, thought to have or at risk of having apathogenic infection. Subjects of an unknown infection status can beexamined to determine if they have an infection, for example usingserological tests, physical examination, enzyme-linked immunosorbentassay (ELISA), radiological screening or other diagnostic techniqueknown to those of skill in the art. In some examples, subjects arescreened to identify a particular pathogen of interest, with aserological test, or with a nucleic acid probe specific for a pathogenof interest, or even a panel of nucleic acid probes, such as an array,that can identify several pathogens simultaneously. Subjects found to(or known to) have a pathogenic infection from a pathogen of interest,for example Streptococcus pneumoniae, Streptococcus pyogenes, Neisseriameningitides or Staphylococcus aureus, such as methicillin resistantStaphylococcus aureus (MRSA), and thereby treatable by administration ofP4 peptide in conjunction with an antibiotic specific for the detectedpathogen of interest are selected for administration of P4 peptide andthe antibiotic specific for the pathogen of interest. Subjects may alsobe selected who are at risk of developing a pathogenic infection forexample, subjects exposed to a known pathogen of interest, the elderly,the immunocompromised and the very young, such as infants.

Subjects selected for treatment are be administered a therapeutic amountof P4 peptide. The P4 peptide can be administered at doses of 1 μg/kgbody weight to about 1 mg/kg body weight per dose, such as 1 μg/kg bodyweight −100 μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kgbody weight per dose, or 500 μg/kg body weight −1000 μg/kg body weightper dose. Subjects are administered a therapeutic amount of opsonicantibody that is specific for the identified pathogen of interest. Theantibiotic can be administered at doses of 1 μg/kg body weight to about1 mg/kg body weight per dose, such as 1 μg/kg body weight −100 μg/kgbody weight per dose, 100 μg/kg body weight −500 μg/kg body weight perdose, or 500 μg/kg body weight −1000 μg/kg body weight per dose.However, the particular dose can be determined by a skilled clinician.The P4 peptide can be administered concurrently or sequentially withantibiotic. The P4 peptide and/or the antibiotic can be administered inone or several doses, for example continuously, daily, weekly, ormonthly. When administered sequentially the time separating theadministration of the P4 peptide and antibiotic can be seconds, minutes,hours, days, or even weeks.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

Example 7 Treatment of Subjects at Risk for Pneumonia with P4 Peptide

This example describes methods that can be used to treat a subject thathas or is at risk of having of pneumonia, for example from a pathogenthat is known to cause pneumonia, such as Streptococcus pneumonia. Inparticular examples, the method includes screening a subject having,thought to have or at risk of having a pathogenic infection that causespneumonia, such as pneumococcal disease, for example infection withStreptococcus pneumonia. Subjects may be selected who are at risk ofdeveloping a pathogenic infection for example, subjects exposed to aknown pathogen of interest, the elderly, the immunocompromised (forexample those on immunosupressive therapies or infected with HIV) andthe very young, such as infants. An increased risk of pneumococcalinfection, such as infection with Streptococcus pneumoniae can beassociated with defects in the non-specific and specific defensemechanisms against colonization, aspiration or invasion by Streptococcuspneumoniae. Examples of such defects include decreased cough reflex,poor cilliary function, and immune deficiencies such ashypogammaglobulinemia, complement defects, leukopenia, or asplenia.Other risk factors include dementia, seizure disorders, current tobaccouse, such as cigarette use, alcohol use, congestive heart failure,cereberovascular disease, institutionalization, and chronic obstructivepulmonary disease (COPD). In subjects with asplenia for example the riskof invasive pneumococcal disease is about 500 per 100,000 per year.

Subjects selected for treatment can be administered a therapeutic amountof P4 peptide. The P4 peptide can be administered at doses of 1 μg/kgbody weight to about 1 mg/kg body weight per dose, such as 1 μg/kg bodyweight −100 μg/kg body weight per dose, 100 μg/kg body weight −500 μg/kgbody weight per dose, or 500 μg/kg body weight −1000 μg/kg body weightper dose. In some examples, subjects are also administered a therapeuticamount of opsonic antibody that is specific for the identified pathogenof interest. The opsonic antibody can be administered at doses of 1μg/kg body weight to about 1 mg/kg body weight per dose, such as 1 μg/kgbody weight −100 μg/kg body weight per dose, 100 μg/kg body weight −500μg/kg body weight per dose, or 500 μg/kg body weight −1000 μg/kg bodyweight per dose. However, the particular dose can be determined by askilled clinician. The P4 peptide can be administered concurrently orsequentially with opsonic antibody. The P4 peptide and/or the opsonicantibody can be administered in one or several doses, for examplecontinuously, daily, weekly, or monthly. When administered sequentiallythe time separating the administration of the P4 peptide and opsonicantibody can be seconds, minutes, hours, days, or even weeks.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used, andit is intended that the disclosure may be practiced otherwise than asspecifically described herein. Features, characteristics, compounds,chemical moieties, or examples described in conjunction with aparticular aspect, embodiment, or example of the invention are to beunderstood to be applicable to any other aspect, embodiment, or exampleof the invention. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims.

We claim:
 1. A method of enhancing opsonophagocytosis of a pathogen ofinterest in a subject, comprising: selecting a subject for treatmentthat has, or is at risk for developing, an infection by a pathogen ofinterest; administering to a subject a therapeutically effective amountof an isolated P4 peptide comprising the amino acid sequence set forthas SEQ ID NO: 1; and administering to the subject a therapeuticallyeffective amount of one or more isolated opsonic antibodies or afragment thereof that specifically binds to an antigen present on thesurface of the pathogen of interest, thereby enhancing theopsonophagocytosis of a pathogen of interest.
 2. The method of claim 1,wherein the pathogen of interest does not express pneumococcal surfaceadhesin A (PsaA) protein
 3. The method of claim 1, wherein the P4peptide consists of the amino acid sequence set forth as SEQ ID NO: 1.4. The method of claim 1, further comprising administering to thesubject a therapeutically effective amount of an antibiotic for thepathogen of interest.
 5. The method of claim 1, further comprisingadministering to the subject a therapeutically effective amount ofisolated complement protein or fragment thereof.
 6. The method of claim5, wherein the complement protein fragment complement is one or more ofC3a, C3b, iC3b, C3d, C4b, or C5a.
 7. The method of claim 1, wherein theisolated P4 peptide is administered by one or more of an intranasalroute, an intravenous route, a topical route, an enteral route, aparenteral route, or a intravitreal route.
 8. The method of claim 1,wherein the pathogen of interest is a bacterial pathogen of interest, aviral pathogen of interest, viral infected cells of interest, or afungal pathogen of interest.
 9. The method of claim 8, wherein thepathogen of interest is a bacterial pathogen comprising Streptococcuspneumoniae, Neisseria meningitides, Streptococcus pyogenes, orStaphylococcus aureus.
 10. A method of enhancing opsonophagocytosis of apathogen of interest that does not express pneumococcal surface adhesinA (PsaA) protein in a subject, comprising: selecting a subject fortreatment that has, or is at risk for developing, an infection by apathogen of interest that does not express PsaA protein; andadministering to the subject a therapeutically effective amount of anisolated P4 peptide comprising the amino acid sequence set forth as SEQID NO: 1, thereby enhancing the opsonophagocytosis of a pathogen ofinterest.
 11. The method of claim 10, further comprising administeringto the subject a therapeutically effective amount of one or moreexogenous isolated opsonic antibodies or a fragment thereof thatspecifically binds to an antigen present on the surface of the pathogenof interest.
 12. The method of claim 10, wherein the P4 peptide consistsof the amino acid sequence set forth as SEQ ID NO:
 1. 13. The method ofclaim 10, further comprising administering to the subject atherapeutically effective amount of an antibiotic for the pathogen ofinterest.
 14. The method of claim 10, further comprising administeringto the subject a therapeutically effective amount of isolated complementprotein or fragment thereof.
 15. The method of claim 14, wherein thecomplement protein fragment complement is one or more of C3a, C3b, iC3b,C3d, C4b, or C5a.
 16. The method of claim 10, wherein the isolated P4peptide is administered by one or more of an intranasal route, anintravenous route, a topical route, an enteral route, a parenteralroute, or a intravitreal route.
 17. The method of claim 10, wherein thepathogen of interest is a bacterial pathogen of interest, a viralpathogen of interest, viral infected cells of interest, or a fungalpathogen of interest.
 18. The method of claim 17, wherein the pathogenof interest is a bacterial pathogen comprising Streptococcus pneumoniae,Neisseria meningitides, Streptococcus pyogenes, or Staphylococcusaureus.
 19. A therapeutic composition comprising, a therapeuticallyeffective amount of an isolated P4 peptide comprising the amino acidsequence set forth as SEQ ID NO: 1; and a therapeutically effectiveamount of one or more exogenous isolated opsonic antibodies or afragment thereof that specifically binds to an antigen present on thesurface of a pathogen of interest.
 20. The therapeutic composition ofclaim 19, wherein the P4 peptide consists of the amino acid sequence setforth as SEQ ID NO:
 1. 21. The therapeutic composition of claim 19further comprising a therapeutically effective amount of an antibioticfor the pathogen of interest.
 22. The therapeutic composition of claim19, further comprising a therapeutically effective amount isolatedcomplement protein or fragment thereof.
 23. The composition of claim 19,wherein the pathogen of interest is a bacterial pathogen of interest, aviral pathogen of interest, viral infected cells of interest, or afungal pathogen of interest.
 24. The composition of claim 23, whereinthe pathogen of interest is a bacterial pathogen comprisingStreptococcus pneumoniae, Neisseria meningitides, Streptococcuspyogenes, or Staphylococcus aureus.
 25. The composition of claim 19,wherein the composition is formulated for intranasal, intravenous,topical, enteral, parenteral, or intravitreal administration.